- Short report
- Open Access
Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes
- Susanne Eriksen Boonen†1,
- Andrea Freschi†2,
- Rikke Christensen1,
- Federica Maria Valente2,
- Dorte Launholt Lildballe1,
- Lucia Perone3,
- Orazio Palumbo4,
- Massimo Carella4,
- Niels Uldbjerg5,
- Angela Sparago2,
- Andrea Riccio2, 6Email author and
- Flavia Cerrato2Email authorView ORCID ID profile
© The Author(s). 2016
Received: 7 April 2016
Accepted: 8 June 2016
Published: 16 June 2016
The overgrowth-associated Beckwith-Wiedemann syndrome (BWS) and the undergrowth-associated Silver-Russell syndrome (SRS) are characterized by heterogeneous molecular defects affecting a large imprinted gene cluster at chromosome 11p15.5-p15.4. While maternal and paternal duplications of the entire cluster consistently result in SRS and BWS, respectively, the phenotypes associated with smaller duplications are difficult to predict due to the complexity of imprinting regulation. Here, we describe two cases with novel inherited partial duplications of the centromeric domain on chromosome 11p15 associated with contrasting growth phenotypes.
In a male patient affected by intrauterine growth restriction and postnatal short stature, we identified an in cis maternally inherited duplication of 0.88 Mb including the CDKN1C gene that was significantly up-regulated. The duplication did not include the long non-coding RNA KCNQ1OT1 nor the imprinting control region of the centromeric domain (KCNQ1OT1:TSS-DMR or ICR2) in which methylation was normal. In the mother, also referring a growth restriction phenotype in her infancy, the duplication was de novo and present on her paternal chromosome. A different in cis maternal duplication, 1.13 Mb long and including the abovementioned duplication, was observed in a child affected by Tetralogy of Fallot but with normal growth. In this case, the rearrangement also included most of the KCNQ1OT1 gene and resulted in ICR2 loss of methylation (LOM). In this second family, the mother carried the duplication on her paternal chromosome and showed a normal growth phenotype as well.
We report two novel in cis microduplications encompassing part of the centromeric domain of the 11p15.5-p15.4 imprinted gene cluster and both including the growth inhibitor CDKN1C gene. Likely, as a consequence of the differential involvement of the regulatory KCNQ1OT1 RNA and ICR2, the smaller duplication is associated with growth restriction on both maternal and paternal transmissions, while the larger duplication, although it includes the smaller one, does not result in any growth anomaly.
Our study provides further insights into the phenotypes associated with imprinted gene alterations and highlights the importance of carefully evaluating the affected genes and regulatory elements for accurate genetic counselling of the 11p15 chromosomal rearrangements.
Less than 1 % of human genes are imprinted, that is, their expression is monoallelic and parent of origin-dependent as a result of epigenetic modifications acquired during gametogenesis . Alterations of imprinted gene expression result in imprinting disorders (IDs) that are characterized by growth, metabolic, and developmental anomalies. Imprinted genes are generally organized in clusters that share regulatory cis-acting elements, such as enhancers and imprinting control regions (ICRs). The ICRs are 2–4-kb long genomic sequences characterized by repressive and permissive epigenetic marks on the opposite parental alleles. A large cluster of imprinted genes that is located on chromosome 11p15.5-p15.4 harbors two independent ICRs, H19/IGF2:IG (Intergenic)-DMR (also known as ICR1), and KCNQ1OT1:TSS (transcription start site)-DMR (also known as ICR2). ICR2 controls the imprinting of the centromeric domain. This region corresponds to the promoter of KCNQ1OT1, a long non-coding RNA that is transcribed antisense to KCNQ1 and represses in cis the flanking imprinted genes on the paternal chromosome. These include KCNQ1, a member of the potassium channel KQT-family, and two genes with growth inhibitory properties, CDKN1C and PHLDA2 [1–3].
Opposite genetic and epigenetic anomalies of the 11p15.5-p15.4 region result in the overgrowth-associated Beckwith-Wiedemann syndrome (BWS, MIM #130650)  and the undergrowth-associated Silver-Russell syndrome (SRS, MIM #180860) . The BWS patients usually show one of the following defects: (1) gain of methylation (GOM) of ICR1 (5–10 % of the cases); (2) loss of methylation (LOM) of ICR2 (50 % of the cases); and (3) aberrant methylation of both ICRs due to segmental paternal uniparental disomy (UPD, 20 % of the cases) of chromosome 11. Conversely, the SRS patients frequently show ICR1 LOM (50 % of the cases); maternal UPD of chromosome 11p15 has been reported in only one case [1, 6]. CDKN1C variations affecting CDKN1C function can also cause these diseases. Maternally inherited loss-of-function mutations have been described in 5 % of the BWS patients (and 50 % of the familial cases) while gain-of-function mutations have been reported in the intrauterine growth restriction (IUGR)-associated IMAGe syndrome and in a single familial case of SRS [7, 8].
Deletions/duplications of chromosome 11p15.5-p15.4 have generally been reported in only 2–6 % of BWS and SRS patients , but a more recent study demonstrates an 8.4 % frequency in BWS patients . Duplications encompassing the entire imprinted gene cluster are usually associated with BWS if paternally inherited and with SRS if maternally inherited. In addition, paternal duplication of the telomeric domain usually results in BWS [11, 12] and maternal duplication of the centromeric domain results in SRS [13, 14]. The contrasting phenotypes observed on maternal and paternal transmission of these chromosome alterations are likely caused by opposite deregulation of IGF2 in the telomeric domain and CDKN1C and PHLDA2 in the centromeric domain . In the case of smaller duplications encompassing only a part of a single domain, the clinical outcome is difficult to predict because of the complex regulation of the 11p15 imprinted gene cluster.
Here, we describe two novel submicroscopic in cis duplications including part of the centromeric domain of the 11p15 imprinted gene cluster. The duplications extend 0.88 and 1.13 Mb from the middle of the KCNQ1 gene toward the centromere, respectively. Despite both chromosome aberrations involve the CDKN1C gene and two out of the three putative enhancers , we find that only the smaller one is associated with growth restriction. The finding that the larger duplication also includes a hypomethylated ICR2 and part of KCNQ1OT1 provides a possible explanation for the associated contrasting growth phenotypes.
The proband of family 1 was the third son of three children from unrelated parents. Pes equinovarus was observed by ultrasound scan by gestational age 16 + 2 weeks and an amniocentesis was obtained. The maternally inherited 11p15 duplication was identified. Due to this finding several ultrasound scans were performed during the pregnancy and IUGR was observed: −18 % at 30 + 6 weeks of gestation, −25 % at 32 + 5 weeks, −27 % at 34 + 5 weeks, and −30 % at 36 + 5 weeks. Due to IUGR induction of delivery was performed. He was born small for gestational age (SGA) at gestational age (GA) 37 + 6 weeks. His birth weight was 2070 g, (−3 SDS), birth length 44 cm (−3 SDS), and occipital frontal circumference (OFC) 30 cm (−2.5 SDS). The placenta weight (300 g, <third centile) was also reduced. Further, blood glucose was 1.4 mmol/l by delivery. He received treatment with intravenous glucose for 1 day. Afterwards he was only breast feed. The pes equinovarus was treated with plaster and tenotomy of the Achilles tendons at 45 days of age.
Physical examination at 14 months confirmed the growth restriction, in particular the short stature: weight 9.0 kg (−1.5 SDS), length 73 cm (−2.5 SDS), and OFC 45 cm (−1.5 SDS). The father, aged 40 years, was referred to be of normal stature (176 cm). The mother’s height was 163 cm and her weight is 59 kg at 38 years of age. It was referred that in childhood, she was very small and the general practitioner suspected she was a kind of a dwarf at 5–6 years of age.
The first son was born by GA 42 weeks with a birth weight of 3320 g (−0.5 SDS) and a birth length of 52 cm (average). By 14 years of age, his height was 168 cm (+1 SDS); the second son was born at 33 weeks of gestation. The spontaneous preterm delivery was caused by membrane rupture. His birth weight was 1575 g (−1.1 SDS), the birth length 41 cm (−1 SDS). Information on fetal growth parameters during pregnancy was not available. He was treated with intravenous glucose infusions age 1–3 days due to low blood glucose (blood glucoses at day 1: 1.9–2.6 mmol/l). By 7 years, his height was 127 cm (+1 SDS). He was affected by mild attention deficit hyperactivity disorder (ADHD).
To confirm the duplication and determine if it was present in cis or in trans, cells of the umbilical cord of the proband were analyzed by fluorescence in situ hybridization (FISH). A bacterial artificial chromosome (BAC) probe (RP11-11A9, chr11: 3236552-3356012, green signal in Additional file 4: Figure S4) hybridizing within the duplicated region and a BAC probe (RP11-876C12, chr11q22.3, red) located outside the duplication were used for the metaphase FISH (Additional file 4: Figure S4, top panel). FISH on interphase nuclei was performed by using the BAC clones RP11-11A9 (green) and RP11-81 K4 (red), both located within the duplicated region (Additional file 4: Figure S4, bottom panel). The absence of signals in chromosomes other than chromosome 11 in metaphase FISH, and the green-red-red-green sequence of the fluorescence signals, demonstrated the presence of an in cis duplication with inverted orientation.
To investigate the effect of the duplication on genomic imprinting, we analyzed the DNA methylation of ICR1 and ICR2 in the placenta cells of the proband by Pyrosequencing (Fig. 2b) and combined bisulfite restriction assay (COBRA; Additional file 5: Figure S5). With both methods, the proband showed a methylation profile comparable to that of three healthy controls in both ICR1 and ICR2. Normal ICRs methylation was also observed in the blood leukocytes of the parents. To look for a possible deregulation of the 11p15 imprinted genes, we analyzed the RNA levels of CDKN1C and PHLDA2 in placenta cells. We found that CDKN1C expression was increased 10-fold (P < 0.01; Fig. 2c) and PHLDA2 threefold in the proband when compared with three healthy controls (P < 0.01; Fig. 2c).
The male proband was the only child of non-consanguineous healthy parents. He was born by GA 37 + 5 weeks. Birth weight was 3350 g (−0.5 SDS), birth length 51 cm (average), and OFC 35 cm (+0.5 SDS). Apgar scores were 8/1, 8/5, and 8/10. Neonatal plasma glucose was normal. In the medical record, it is described that there was slight cranial asymmetry with left side of parietal and frontal region a little flat. The head was described as slight narrow, the nasal bridge as slightly wide, and there was strabismus and retention testis. Further, there were described bilateral dysplastic nails on third, fourth, and fifth toes. He was affected by Steno-Fallot Tetralogy, diagnosed on day 1 by echocardiography required because of a systolic murmur. Operation was performed by age 8 month. Neonatal ultrasound scans of cerebrum and kidneys were both normal. At 1 year old, he showed slight frontal bossing, slight hypoplasia of maxilla, slightly flaccid occiput, and bilateral single palmar creases. Neither umbilical hernia nor ear lobe creases were observed. At 8 and 20 months of age, the auxological parameters were still close to the average: 8 month: weight 9.2 kg (+0.5 SDS), length 71.5 cm (+0.5 SDS) and OFC 44 cm (−0.5 SDS); 20 month: weight 11.3 kg (−0.5 SDS), length 84.5 cm (−0.5 SDS) and OFC 46.3 cm (−1.5 SDS). The psychomotor development was normal.
The mother was 35 years old, with normal phenotype except for the presence of bilateral ear lobe creases. Her height was 170 cm and weight was 56.5 kg. She was born by GA 41 + 5 weeks, with the birth weight 3740 g (+0.5 SDS), birth length 53 cm (+1 SDS), and OFC 35.5 cm (+1.5 SDS).
Copy number and DNA methylation of the chromosome 11p15.5 region were first analyzed by MS-MLPA. Increased hybridization signal at ICR2 and KCNQ1 exons 13–17 and slight loss of ICR2 methylation were identified in the proband and his mother, while ICR1, IGF2, H19, and control probes showed normal copy number and methylation status (Additional file 6: Figure S6), indicating the presence of an inherited partial duplication of the 11p15.5-p15.4 imprinted gene cluster.
To better define the DNA methylation abnormality of the 11p15.5-p15.4 region in the proband and his mother, the methylation levels of the ICRs were determined by pyrosequencing in the trio. As shown in Fig. 2d, the methylation profiles of both ICRs were normal in the father, while the proband and his mother showed normal methylation of ICR1 but hypomethylation of ICR2 at a level comparable with other previously described ICR2 duplication carriers [17, 18]. The allele-specific methylation analysis could not be performed because of the absence of polymorphisms in the ICR2 sequence. Nevertheless, the observed hypomethylation suggests that the duplicated ICR2 fails to acquire or maintain the maternal imprints in the proband.
The inheritance of the duplicated region in the proband, his parents and maternal grandparents, was determined by analyzing the 11p15 microsatellite markers. The segregation and signal intensity of the D11S4088 marker, located in the duplicated region, confirmed that the duplication was maternally inherited in the proband and demonstrated that it originated de novo from the paternal chromosome in the mother (Fig. 2e and Additional file 7: Figure S7). The markers, D11S4046, D11S922, and TH, did not show any allelic imbalance in the proband and his mother, consistent with their localization outside of the duplicated region (Additional file 7: Figure S7).
To determine the chromosomal location of the duplicated region, the cultured blood leukocytes of the proband were analyzed by FISH. The BAC probes hybridizing within the duplicated region, RP11-11A9 (chr11: 3236552-3356012, green) and RP11-699D10 (chr11: 2.9–3.04 Mb, red), were used. As in family 1, the results of the metaphase FISH indicated that the duplication was in cis (Additional file 8: Figure S8, top panel), while the FISH on interphase nuclei demonstrated the inverted orientation of the duplication (Additional file 8: Figure S8, bottom panel).
Maternal duplications of the centromeric domain of the 11p15 imprinted gene cluster generally result in SRS phenotype. In this study, we describe two familial cases with overlapping maternal duplications that partially affect the centromeric domain and show contrasting growth phenotypes.
Both the probands mothers are carriers of the duplications but on their paternal chromosomes. The family 1 mother (II-2) was growth restricted during her infancy. This phenotype likely results from CDKN1C expression on both maternal and paternal 11p15 chromosomes (Fig. 3). No CDKN1C deregulation is expected instead in the mother of family 2 because of the KCNQ1OT1 duplication and ICR2 hypomethylation.
Maternal duplication of the entire centromeric domain or the entire 11p15 imprinted gene cluster is generally associated with clinical features of SRS and quite severe growth restriction (birth weight and length z scores −2.5/−7 SDS; postnatal growth restriction −2.5/−6.4 SDS; ). In contrast, the family 1 proband showed limited growth restriction and no other characteristics of SRS. Also, the occurrence of a compensatory growth later in development (he was 14 months old at the last examination) cannot be excluded. An even milder phenotype was observed in the second brother (III-2) of the proband, who is also a carrier of the duplication. He was of short stature in the first 3 years of life (height around 20–25th centile: 41 cm at birth, 65 cm (−1.5 SDS) at 6 months, 89 cm at 29 months (−0.5 SDS)), had hypoglycemia at birth, and was affected by ADHD. The attenuated phenotypes may be due to the limited extension of the 0.88 Mb duplication into the centromeric domain leaving out some of the putative CDKN1C enhancers (Fig. 3). It is worth to mention, however, that a mild SRS-like phenotype with ADHD was recently reported associated with a 1.9 Mb maternal duplication encompassing the entire 11p15.5 cluster .
Only a few other duplications encompassing partially the centromeric domain of the 11p15 imprinted gene cluster have been described so far. Two of these are 50 and 160 kb long, respectively, and both were associated with BWS upon maternal transmission. The 50-kb duplication spans from intron 1 to intron 2 of KCNQ1 and was associated with ICR2 hypomethylation . The 160 kb duplication spans from intron 9 to exon 15 of KCNQ1 and included a non-methylated copy of ICR2 and the 5′ part of the KCNQ1OT1 gene . In both cases, the BWS phenotype likely results from the expression of the maternal KCNQ1OT1 allele causing CDKN1C repression in cis. A complex 277 Kb rearrangement on the paternal chromosome 11p15 has been recently described associated with SRS . In this case, a small portion of KCNQ1OT1 and the entire CDKN1C gene were duplicated, but these duplications were discontinuous and did not include ICR2. In this case, the SRS phenotype likely results from the unregulated expression of CDKN1C on the paternal chromosome.
The 1.13-Mb duplication described in this study has some similarities with the 160 kb duplication associated with BWS . Both rearrangements are present in cis and in inverted orientation and include a duplicated incomplete but likely functional copy of KCNQ1OT1 and a hypomethylated ICR2. However, the former rearrangement is more extended toward the centromere and includes CDKN1C. The consequence is that the 160 kb duplication results in reduced CDKN1C expression and BWS, while the 1.13-Mb duplication is associated with normal growth and likely normal CDKN1C level (Fig. 3).
The Tetralogy of Fallot (TOF) affecting the proband of family 2 is a severe congenital heart malformation (MIM #187500) with both environmental and genetic etiology. The genetics of TOF is complex and involves many loci, but studies performed on large cohorts have not identified any strict association with defects on chromosome 11p [21, 22]. Nevertheless, a few cases of paternal 11p15 duplications with BWS and cardiac malformations including TOF have been described [23–25]. Therefore, the involvement of 11p15 genes in the etiology of rare TOF cases deserves further investigations.
Ear lobe creases are a common sign of BWS . Although more frequently associated with CDKN1C alterations, they can be found in BWS cases with other types of 11p15.5 molecular defect including paternal duplications. The finding of such sign in the mother of family 2 proband is intriguing and may be due to altered expression of some 11p15.5 genes or may be coincidental and have different causes.
In summary, our study is an example of how the analysis of the small copy number variation (CNVs) affecting the imprinted gene clusters can increase our understanding on the imprinting regulatory mechanisms and help to predict the clinical phenotypes resulting from such type of rearrangements.
DNA from peripheral blood leukocytes of all the family members was extracted by an automated Chemagic Magnetic Separation Module (PerkinElmer, Waltham, MA, USA). DNA from buccal swab samples of the relatives of the proband from family 1 was extracted by using the Maxwell 16 LEV Buccal Swab DNA kit (Promega, Madison, WI, USA).
Cells from umbilical cord and placenta of the proband from family 1 and three normal controls were cultured in BIOAMFTM-3 (BI-USA Inc., Cromwell, CT, USA). DNA from cell culture of placenta and umbilical cord and amniotic fluid cells was extracted directly using the Maxwell 16 LEV Blood DNA kit (Promega, Madison, WI, USA). RNA was extracted by using TRIzol reagent (Life technologies, Darmstadt, Germany), according to the protocol of the manufacturer.
All the genetic analyses were performed after informed consent had been obtained. All the clinical and research have been done following the ethical rules of the Danish and Italian law.
Z scores of each member under study were calculated referring to standard growth rate of Denmark.
Copy number variation detection
MS-MLPA. MS-MLPA was performed on genomic DNA of the proband from family 2, his parents and maternal grandparents. The SALSA MS-MLPA kit ME030-C3 for BWS–SRS (MRC-Holland, Amsterdam, the Netherlands) was used following the manufacturer’s instructions. The amplified products were separated by capillary electrophoresis using ABI 3130 Genetic Analyzer (Applied Biosystems, CA, USA). Data was analyzed using the built-in MS-MLPA tool of the software Genemarker v 2.2.0 (Softgenetics, USA).
The samples were analyzed using the SurePrint G3 Human CGH microarray 180k (Agilent Technologies Inc., Santa Clara, CA, USA). Sample and reference genomic DNA (500 ng) were labelled with Cy5 (reference) or Cy3 (specimen) using the Sure Tag Complete DNA labelling Kit (Agilent Technologies Inc.) and purified as described in the manufacturer’s protocol. Labelled sample and reference DNA were pooled, and 5-μl human COT-1 DNA (1 mg/ml), 10× blocking agent, and 2× hybridization buffer were added. Hybridization was performed for 24 h at 65 °C. Scanning and image acquisition were carried out using an Agilent microarray scanner and microarray image files were analyzed using CytoGenomics, version 2.9 (Agilent Technologies Inc.). Copy number was determined using the adm-2 algorithm and profile deviations consisting of four or more neighboring oligonucleotides were considered genomic aberrations. The resolution is thus approximately 50 kb.
Detected copy number gains or losses were compared with our in-house database of CNVs and with public CNV databases (Database of Genomic Variants: http://dgv.tcag.ca/dgv/app/home; Decipher: http://decipher.sanger.ac.uk; ISCA: http://clinicalgenome.org/).
Whole-genome copy number variation (CNV) analysis was carried out using the CytoScan HD array platform (Affymetrix, Santa Clara, CA). This array contains more than 2.6 million markers for copy number analysis and approximately 750,000 SNPs that fully genotype with greater than 99 percent accuracy. The CytoScan HD assay was performed starting with 250 ng DNA as previously described . Both quality control step and copy number analysis were performed using the Chromosome Analysis Suite Software version 2.0. The raw data file (.CEL) was normalized using the default options; an unpaired analysis was performed using as baseline 270 HapMap samples in order to obtain copy numbers value from .CEL files; while the amplified and/or deleted regions were detected using a standard Hidden Markov Model (HMM) method. Karyotype was designated according to ISCN 2013, and base pair position was derived from the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway), build GRCh37 (hg19).
D11S4088 short tandem repeat (STR) marker mapping to the duplicated region and TH, D11S4046 and D11S900 STR mapping at the 11p15.5-4 region outside the duplication, were analyzed in the probands and their relatives to verify the origin of duplication and follow the segregation through the three generations. Primers specific for the STR were obtained from NCBI Genome Database together with the PCR conditions. PCR amplification of 100-ng DNA was done using forward primer end labelled with Fam or Hex. Twenty-eight cycles of PCR products were run on the fluorescent capillary system ABI 3130XL. Data were analyzed using GeneMapper Software.
Microsatellites of chromosome 7 (D7S657, D7S502, D7S686, D7S1830) were also analyzed to exclude the UPD7 associated with the 10 % of the SRS cases (data not shown).
Fluorescence in situ hybridization
FISH analysis was performed on metaphase or interphase nuclei spread from PHA-stimulated umbilical cord cell culture (proband, family 1) and peripheral blood leukocytes (proband, family 2) using standard procedures. The RP11-699D10 (red) and RP11-11A9 (green) BlueFISH probes (Illumina) targeting the 11p15.4 duplicated region were used for FISH analyses of proband of family 1. RP11-179B7 (red) on 11q22.3 served as control for chromosome 11 to exclude a translocation defect. RP11-11A9 (green) and the BAC clone RP11-81 K4 DNA labelled with red fluorophore using a non-enzymatic nucleic acid labelling method (ULSTM, Kreatech Diagnostics Amsterdam, The Netherlands) were used as probes for the interphase FISH on proband of family 2. The probes used for the metaphase FISH were RP11-81K4 (11p15.5-15.4, green) and RP11-876C12 (11q22.3, red). The chromosomes and nuclei were counterstained with DAPI. Hybridizations were analyzed using a Leica DMRB microscope (Leica Microsystems A/S, Wetzlar, Germany) or a Nikon Eclipse-1000 epifluorescence microscope (Nikon Instruments, Tokyo, Japan). Images captured and elaborated using the ISIS software v. 5.1 (MetaSystems GmbH, Altlussheim, Germany) or the Genikon systemv. 3.8.5 (Nikon Instruments, Tokyo, Japan).
DNA methylation analysis
Two micrograms of genomic DNA extracted from cells/tissues was treated with sodium bisulfite by using the EpiTect Bisulfite kit (Qiagen-Italia, Milan, Italy) following the manufacturer’s protocol. The converted DNA was analyzed by COBRA and Pyrosequencing.
Bisulfite-treated DNA was amplified with primers specific for CTCF target site 1 of ICR1 and ICR2. The PCR products were then digested with BstUI (CGCG) and the digestion products were run on a polyacrylamide gel to separate the digested (methylated) from the undigested (non-methylated) bands. The percentage of methylation was calculated by computer quantitation of the gel following exposure to phosphorimager. Primers sequences, PCR, and restriction enzyme reaction conditions were previously described [27, 28].
In order to obtain more quantitative DNA methylation data, pyrosequencing was performed to assess methylation at seven CpGs within ICR2 and three CpGs within ICR1, as control. Primers and PCR conditions were previously described  (KvDMR1-F 5′-TTAGTTTTTTGYGTGATGTGTTTATTA-3′ and KvDMR1-R 5′-Biotin/CCCACAAACCTCCACACC-3′; for sequencing: KvDMR1-S 5′-TTGGTAGGTATAGAAATTGGGG-3′) and  (H19DMR-CTCF3 F 5′-TTGGTAGGTATAGAAATTGGGG-3′ and H19DMR-CTCF3R 5′-Biotin/ACACYTAACTTAAATAAC-3′; for sequencing: H19DMR-CTCF3 S2 5′-GTGGATTTAAAAGTGGT-3′). Sequencing of 10 μl of PCR product was carried out on a PSQ 96MD system with the PyroGold SQA Reagent Kit (Qiagen-Italia, Milan, Italy), and results were analyzed using the Q-CpG software (V.1.0.9Pyrosequencing).
Gene expression analysis
About 1 μg of total RNA extracted from placenta and umbilical cord cultured cells was treated with RNase-free DNase, and first-strand complementary DNA (cDNA) was synthesized using Quantitech Reverse Transcription Kit (Qiagen-Italia, Milan, Italy), according to the protocol of the manufacturer. CDKN1C expression was examined by SYBR Green quantitative real-time PCR (Power SYBR Green Master Mix Applied Biosystems, Foster City, CA, USA). Reactions were run on ABI PRISM 7500 using the default cycling conditions. Relative expression was determined using the ΔΔC T method, and gene expression values were normalized to the expression of the GAPDH reference gene. The primers used are CDKN1C For 5′- AGAGATCAGCGCCTGAGAAG-3′ and CDKN1C Rev 5′-CACCTTGGGACCAGTGTACC-3′ ; GAPDH For 5′-CACCATCTTCCAGGAGCGAG-3′ and GAPDH Rev 5′-TCACGCCACAGTTTCCCGGA-3′.
BAC, bacterial artificial chromosome; BWS, Beckwith-Wiedemann syndrome; CGH, array comparative genomic hybridization; COBRA, combined bisulfite restriction assay; Ctrl, control; Dupl, duplication; FISH, fluorescence in situ hybridization; GOM, gain of methylation; ICR, imprinting control region; ID, imprinting disorders; IG-DMR, intergenic differentially methylated region; LOM, loss of methylation; MS-MLPA, methylation-specific multiplex-ligation-dependent amplification probe assay; OFC, occipital frontal circumference; SDS, standard deviation score; SNP, single nucleotide polymorphisms; SRS, Silver-Russell syndrome; TSS-DMR, transcription start site—differentially methylated region; UPD, uniparental disomy
We thank the patients and the patients’ families for their collaboration and Alice Nielsen and Inge-Lise Floor for the excellent technical assistance. This research was supported by Telethon by grants from Telethon-Italia grant n. GGP15131 (AR), Associazione Italiana Ricerca sul Cancro, grant IG 2012 N.12815 (AR), Progetto Bandiera MIUR-CNR Epigenomica (AR), and EU-FP7-ITN INGENIUM n. 290123 (AR).
AF, FMV, and AS carried out the molecular genetic studies of the patients. LP carried out the FISH analysis. OP and MC performed the SNP array analysis. SEB, RC, and DLL identified the patients, performed the CGH array, metaphase FISH, and MS-MLPA and contributed to the interpretation of results. NU provided the normal placenta cells. The clinical studies were performed by SEB. FC and AR carried out study design, data analysis, interpretation, manuscript writing, and revision. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Eggermann T, Perez de Nanclares G, Maher ER, Temple IK, Tümer Z, Monk D, Mackay DJ, Grønskov K, Riccio A, Linglart A, Netchine I. Imprinting disorders: a group of congenital disorders with overlapping patterns of molecular changes affecting imprinted loci. Clin Epigenetics. 2015;7:123.View ArticlePubMedPubMed CentralGoogle Scholar
- Eggermann T, Binder G, Brioude F, Maher ER, Lapunzina P, Cubellis MV, et al. CDKN1C mutations: two sides of the same coin. Trends Mol Med. 2014;20:614–22.View ArticlePubMedGoogle Scholar
- Ishida M, Monk D, Duncan AJ, Abu-Amero S, Chong J, Ring SM, et al. Maternal inheritance of a promoter variant in the imprinted PHLDA2 gene significantly increase birth weight. Am J Hum Genet. 2012;90:715–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Choufani S, Shuman C, Weksberg R. Molecular finding in Beckwith-Wiedemann syndrome. Am J Med Genet C Semin Med Genet. 2013;163:131–40.View ArticleGoogle Scholar
- Eggermann T. Russell-Silver syndrome. Am J Med Genet C Semin Med Genet. 2010;154:355–64.View ArticleGoogle Scholar
- Bullman H, Lever M, Robinson DO, Mackay DJ, Holder SE, Wakeling EL. Mosaic maternal uniparental disomy of chromosome 11 in a patient with Silver-Russel syndrome. J Med Genet. 2008;45:396–9.View ArticlePubMedGoogle Scholar
- Arboleda VA, Lee H, Parnaik R, Fleming A, Banerjee A, Ferraz-de-Souza B, et al. Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome. Nat Genet. 2012;44:788–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Brioude F, Oliver-Petit I, Blaise A, Praz F, Rossignol S, Le Jule M, et al. CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome. J Med Genet. 2013;50:823–30.View ArticlePubMedGoogle Scholar
- Demars J, Rossignol S, Netchine I, Lee KS, Shmela M, Faivre L, et al. New insights into the pathogenesis of Beckwith-Wiedemann and Silver-Russell syndromes: contribution of small copy number variations to 11p15 imprinting defects. Hum Mutat. 2011;32:1171–82.View ArticlePubMedGoogle Scholar
- Baskin B, Choufani S, Chen Y, Shuman C, Parkinson N, Lemyre E, et al. High Frequency of copy number variations (CNVs) in the chromosome 11p15 region in patients with Beckwith-Wiedemann syndrome. Hum Genet. 2014;133:321–30.View ArticlePubMedGoogle Scholar
- Algar EM, St Heaps L, Darmanian A, Dagar V, Prawitt D, Peters GB, Collins F. Paternally inherited submicroscopic duplication at 11p15.5 implicates insulin-like growth factor II in overgrowth and Wilms' tumorigenesis. Cancer Res. 2007;67:2360–5.View ArticlePubMedGoogle Scholar
- Bliek J, Snijder S, Maas SM, Polstra A, van der Lip K, Alders M, Knegt AC, Mannens. Phenotypic discordance upon paternal or maternal transmission of duplications of the 11p15 imprinted regions. Eur J Med Genet. 2009;52:404–8.View ArticlePubMedGoogle Scholar
- Schonherr N, Meyer E, Roos A, Schmidt A, Wollmann HA, Eggermann T. The centromeric 11p15 imprinting centre is also involved in Silver-Russell syndrome. J Med Genet. 2007;44:59–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Bonaldi A, Mazzeu JF, Costa SS, Honjo RS, Bertola DR, Albano LM, et al. Microduplication of the ICR2 domain at chromosome 11p15 and familial Silver-Russell syndrome. Am J Med Genet A. 2011;155A:2479–83.View ArticlePubMedGoogle Scholar
- Begemann M, Spengler S, Gogiel M, Grasshoff U, Bonin M, Betz RC, et al. Clinical significance of copy number variations in the 11p15.5 imprinting control regions: new cases and review of the literature. J Med Genet. 2012;49:547–53.View ArticlePubMedPubMed CentralGoogle Scholar
- Cerrato F, De Crescenzo A, Riccio A. Looking for CDKN1C enhancers. Eur J Hum Genet. 2014;22:442–3.View ArticlePubMedGoogle Scholar
- Chiesa N, De Crescenzo A, Mishra K, Perone L, Carella M, Palumbo O, et al. The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith-Wiedemann syndrome and Silver-Russell syndrome cases. Hum Mol Genet. 2012;21:10–25.View ArticlePubMedGoogle Scholar
- Cardarelli L, Sparago A, De Crescenzo A, Nalesso E, Zavan B, Cubellis MV, et al. Silver-Russell syndrome and Beckwith-Wiedemann syndrome phenotypes associated with 11p duplication in a single family. Pediatr Dev Pathol. 2010;13:326–30.View ArticlePubMedGoogle Scholar
- Brown LA, Rupps R, Peñaherrera MS, Robinson WP, Patel MS, Eydoux P, et al. A cryptic familial rearrangement of 11p15.5, involving both imprinting centers, in a family with a history of short stature. Am J Med Genet A. 2014;164A:1587–94.View ArticlePubMedGoogle Scholar
- Xue Y, Shankar S, Cornell K, Dai Z, Wang C, Rudd MK, et al. Paternal duplication of the 11p15 centromeric imprinting control region is associated with increased expression of CDKN1C in a child with Russell-Silver syndrome. Am J of Med Genet Part A. 2015;167A:3229–33.View ArticleGoogle Scholar
- Greenway SC, Pereira AC, Lin JC, DePalma SR, Israel SJ, Mesquita SM, et al. De novo copy number variants identify new genes and loci in isolated sporadic Tetralogy of Fallot. Nat Genet. 2009;41:931–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Soemedi R, Topf A, Wilson IJ, Darlay R, Rahman T, Glen E, et al. Phenotype-specific effect of chromosome 1q21.1 rearrangements and GJA5 duplications in 2436 congenital heart disease patients and 6760 controls. Hum Mol Genet. 2012;2:1513–20.View ArticleGoogle Scholar
- Slavotinek A, Gaunt L, Donnai D. Paternally inherited duplications of 11p15.5 and Beckwith-Wiedemann syndrome. J Med Genet. 1997;34:819–26.View ArticlePubMedPubMed CentralGoogle Scholar
- Turleau C, de Grouchy J, Chavin-Colin F, Martelli H, Voyer M, Charlas R. Trisomy 11pl5 and Beckwith-Wiedemann syndrome. A report of two cases. Hum Genet. 1984;67:219–21.View ArticlePubMedGoogle Scholar
- Waziri M, Patil SR, Hanson JW, Bartley JA. Abnormality of chromosome 11 in patients with features of Beckwith-Wiedemann syndrome. J Pediatr. 1983;102:873–6.View ArticlePubMedGoogle Scholar
- Palumbo O, Fichera M, Palumbo P, Rizzo R, Mazzolla E, Cocuzza DM, et al. TBR1 is the candidate gene for intellectual disability in patients with a 2q24.2 interstitial deletion. Structure, mechanism, and evolution of the mRNA capping apparatus. Am J Med Genet. 2014;164A:828–33.View ArticlePubMedGoogle Scholar
- Sparago A, Russo S, Cerrato F, Ferraiuolo S, Castorina P, Selicorni A, et al. Mechanisms causing imprinting defects in familial Beckwith-Wiedemann syndrome with Wilms’ tumour. Hum Mol Genet. 2007;16:254–64.View ArticlePubMedGoogle Scholar
- Bliek J, Verde G, Callaway J, Maas SM, De Crescenzo A, Sparago A, et al. Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith-Wiedemann syndrome. Eur J Hum Genet. 2009;17:611–9.View ArticlePubMedGoogle Scholar
- Bourque DK, Avila L, Peñaherrera M, von Dadelszen P, Robinson WP. Decreased placental methylation at the H19/IGF2 imprinting control region is associated with normotensive intrauterine growth restriction but not preeclampsia. Placenta. 2010;31:197–202.View ArticlePubMedGoogle Scholar
- Dejeux E, Olaso R, Dousset B, Audebourg A, Gut IG, Terris B, et al. Hypermethylation of the IGF2 differentially methylated region 2 is a specific event in insulinomas leadingto loss-ofimprinting and overexpression. Endocr Relat Cancer. 2009;16:939–52.View ArticlePubMedGoogle Scholar