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Original research
Paternal 132 bp deletion affecting KCNQ1OT1 in 11p15.5 is associated with growth retardation but does not affect imprinting
  1. Thomas Eggermann1,
  2. Florian Kraft1,
  3. Eva Lausberg1,
  4. Katrin Ergezinger2,
  5. Erdmute Kunstmann3
  1. 1 Institute of Human Genetics, Medical Faculty, RWTH Aachen University, Aachen, Nordrhein-Westfalen, Germany
  2. 2 Children’s Hospital, University of Würzburg, Würzburg, Germany
  3. 3 Institute of Human Genetics, University of Würzburg, Würzburg, Germany
  1. Correspondence to Professor Thomas Eggermann, Institute of Human Genetics, RWTH Aachen University, Aachen D-52074, Germany; teggermann{at}


Background The chromosomal region 11p15.5 harbours two imprinting centres (H19/IGF2:IG-DMR/IC1, KCNQ1OT1:TSS-DMR/IC2). Molecular alterations of the IC2 are associated with Beckwith-Wiedemann syndrome (BWS), whereas only single patients with growth retardation and Silver-Russell syndrome (SRS) features have been reported. CNVs in 11p15.5 account for less than 1% of patients with BWS and SRS, and they mainly consist of duplications of both ICs either affecting the maternal (SRS) or the paternal (BWS) allele. However, this correlation does not apply to smaller CNVs, which are associated with diverse clinical outcomes.

Methods and results We identified a family with a 132 bp deletion within the KCNQ1OT1 gene, associated with growth retardation in case of paternal transmission but a normal phenotype when maternally inherited. Comparison of molecular and clinical data with cases from the literature helped to delineate its functional relevance.

Conclusion Microdeletions within the paternal IC2 affecting the KCNQ1OT1 gene have been described in only five families, and they all include the differentially methylated region KCNQ1OT1:TSS-DMR/IC2 and parts of the KCNQ1 gene. However, these deletions have different impacts on the expression of both genes and the cell-cycle inhibitor CDKN1C. They thereby cause different phenotypes. The 132 bp deletion is the smallest deletion in the IC2 reported so far. It does not affect the IC2 methylation in general and the coding sequence of the KCNQ1 gene. Thus, the deletion is only associated with a growth retardation phenotype when paternally transmitted but not with other clinical features in case of maternal inheritance as observed for larger deletions.

  • imprinting
  • genetics
  • copy-number

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The chromosomal region 11p15.5 harbours two imprinting centres (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) which regulate the monoallelic expression of several imprinted genes. Disturbances of the 11p15.5 imprinted gene clusters are associated with the opposite growth disorders Beckwith-Wiedemann syndrome (BWS; OMIM #130650) and Silver-Russell syndrome (SRS; OMIM #180860), and comprise both genomic alterations (CNVs, point mutations, UPD) as well as methylation defects (epimutations) (for review, see refs 1 2). Whereas patients with BWS exhibit a loss of methylation of the centromeric IC2, gain of methylation of IC1, paternal UPD of 11p15.5 or CNVs in 11p15.5, patients with SRS almost exclusively show hypomethylation of the telomeric IC1.

Among others, the IC1 regulates the expression of the growth-promoting gene IGF2, and due to its monoallelic expression from the paternal allele loss-of-function variants cause growth retardation in case of paternal inheritance.

The IC2 controls the expression of the cell-cycle inhibiting factor CDKN1C, which is maternally expressed and has a clinical impact if the maternal allele is disturbed (figure 1). In contrast to IGF2, CDKN1C mutations can cause both BWS and SRS, either as loss-of-function or gain-of-function variants, respectively. In fact, CDKN1C is indirectly regulated by the IC2 via the non-coding transcript KCNQ1OT1. The antisense KCNQ1OT1 gene (KCNQ1 overlapping transcript 1, LIT1, OMIM #604115) is located in intron 11 of the protein-coding gene KCNQ1, in which pathogenic variants are associated with long QT syndrome (OMIM #192500). Methylation of the IC2 on the maternal allele inhibits KCNQ1OT1 expression, which therefore cannot suppress the transcription of CDKN1C on the same allele. Accordingly, the paternal CDKN1C allele is suppressed by the expression of KCNQ1OT1 on the same, unmethylated allele.

Figure 1

The KCNQ1OT1:TSS-DMR/IC2 in 11p15.5. The paternal unmethylated (empty lollipops) KCNQ1OT1 copy is expressed and thereby suppresses CDKN1C. The maternal allele is methylated (filled lollipops) and therefore suppresses KCNQ1OT1 expression. As a result, CDKN1C is expressed. CDKN1C expression is additionally enhanced by elements on the maternal KCNQ1 allele (green filled circles). A deletion within the paternal KCNQ1OT1 copy (lower panel, right figure) disturbs the non-coding RNA and thereby allows CDKN1C expression on the paternal allele. In case of maternal transmittance, expression of CDKN1C is not altered.

CNVs in 11p15.5 account for less than 1% of patients with BWS and SRS, and they mainly consist of duplications of both ICs with a strict correlation between the parental origin of the duplicated allele and the phenotype (for review, see ref 3). Maternal duplications are associated with SRS features, whereas paternal duplications result in BWS. However, this correlation does not apply to smaller CNVs, which can be associated with unexpected clinical outcomes4 (for review, see refs 3 5). With the molecular characterisation of such rare cases, an increased understanding of the complex regulation of both the IC1 and the IC2 in 11p15.5 has been achieved. Based on IC1 deletions with different sizes and genomic extent, it has become obvious that the presence and spatial order of specific transcription factor binding sites are required for proper methylation and balanced expression of the respective genes.6 7 Methylation of the IC2 has recently been shown to be influenced by the KCNQ1 transcript, as its absence results in failure of IC2 methylation in the female germline.8–11 Thus, in case of maternal inheritance of disrupted KCNQ1 variants, IC2 hypomethylation occurs and is associated with BWS features, but the patients additionally exhibit prolongation of the ECG QT interval (long QT syndrome). The expression of CDKN1C is not only controlled by KCNQ1OT1, but it has been shown that different enhancer motifs within the KCNQ1 region regulate its transcription (for review, see refs 4 8 12 13).

In this paper we report on the smallest deletion within the KCNQ1OT1 gene identified so far, causing a growth retardation phenotype in case of paternal transmission but without consequences for methylation of IC2.

The patient

The girl was the only child of healthy non-consanguineous German parents (mother 159 cm (−0.7 z); father 165 cm (−1.5 z); the target height calculation by Tanner’s formula was 155.5±8.5 cm (−2.04 z)). Family history was empty, and miscarriages were not reported. Pregnancy occurred spontaneously and was uneventful. The patient was born at 35 gestational weeks with a birth weight of 1645 g (−1.95 z), length of 43.5 cm (−1.32 z) and head circumference (occipital frontal circumference, OFC) of 30.5 cm (−1.3 z). In the first week of life, breastmilk was given with a stomach tube, after which a baby bottle was used for nutrition. At the age of 24 months, her height was 78 cm (−2.54 z), weight was 8.26 kg (−2.91 z), body mass index (BMI) was 13.58 kg/m2 (−1.98 z) and OFC was 46 cm (−1.78 z).

At time of first presentation (5 2/12 years), her height was 91.3 cm (−3.98 z), weight was 10.8 kg (−4.57 z), BMI was 13.01 kg/m2 (−1.84 z) and OFC was 48 cm (−1.90 z). Apart from a (mild) prominent forehead, low-set prominent ears and downturned corners of the mouth, dysmorphism or asymmetry was not noted. Cognitive and motor development was normal (figure 2). Clinical scoring for SRS leaned on the recently suggested Netchine-Harbison score revealed a score of only 2 out of 6 parameters (postnatal growth retardation, prominent forehead), but further features were indicative of SRS (low-set, posteriorly rotated ears, facial appearance) and prompted SRS testing.

Figure 2

The patient at the age of 4 10/12 years.

Diagnostic investigations showed delayed bone age (16 months delayed), IGFBP3 of 2.6 µg/mL (1.3–5.6) and IGF1 of 45 µg/L (35.4–232). Growth hormone stimulation test with arginine was normal (peak GH (growth hormone) 8.86 µg/L). Screening laboratory tests ruled out other causes of growth retardation, for example, inflammatory diseases, coeliac disease and hypothyroidism. Karyotype was 46,XX. No pathogenic variant in the SHOX gene was detected (multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing). ECG was normal.

Materials and methods

Genomic DNA of the patient and her parents was isolated from peripheral blood lymphocytes by a simple salting-out procedure. Due to the clinical features reminiscent of SRS, the IC1 and IC2 in 11p15.5 were analysed by two commercially available methylation-specific multiplex ligation-dependent probe amplification kits (MS-MLPA) (ME030-C3 and ME034-A1, MRC Holland, Amsterdam, The Netherlands). The genomic sequence of the region affected by the deletion was amplified by standard PCR and analysed by Sanger sequencing. PCR was carried out according to a standard protocol (20 ngDNA, recombinant Taq Polymerase (Invitrogen, Carlsbad, California, USA), 200 µM dNTPs) with primers covering the affected region (10 µM each; KCNQ1OT1del_F: GAACAGAACCGCGGCGAG; KCNQ1OT1del_R: CCCCTCAGCGCGATTCTG).


In a patient with growth retardation referred for diagnostic testing, a heterozygous deletion of one MLPA probe (KCNQ1OT1-393bp, 07172-L06781) was detected in two MS-MLPA assays. In the methylation-specific analysis the respective probe revealed a hybridisation pattern corresponding to a hypermethylation (figure 3A). This pattern indicated that the paternal allele should be affected by the deletion. The deletion was then confirmed by analysis of the parental DNA samples. Whereas the maternal sample revealed a normal MS-MLPA pattern, the hybridisation of the paternal DNA confirmed the deletion, which was associated with a hypomethylation within the affected region, corresponding to a heterozygous deletion of the maternal allele (figure 3A).

Figure 3

Identification of the 132 bp deletion in the patient and her father. (A) Results of MS-MLPA analysis (upper panel: copy number analysis; lower panel: methylation analysis). The patient and her father exhibit a one probe deletion. In the patient the observed hypermethylation for this probe indicates that the deletion affects the paternal allele, whereas in the father the observed hypomethylation shows that the maternal allele is affected. (B) Sanger sequencing of the breakpoints of the deletion. The position of the affected MLPA probe as well as the breakpoint sequences are indicated. MS-MLPA, methylation-specific multiplex ligation-dependent probe amplification.

By Sanger sequencing of the region, a 132 bp deletion could be identified, affecting the target region of the KCNQ1OT1-393bp probe (probe: 07172-L06781) (figure 3B). The deletion was localised in exon 1 of the non-coding KCNQ1OT1 gene and intron 11 of the KCNQ1 gene (KCNQ1OT1: NR_002728.3:n.134_265del; KCNQ1: LRG287t1:c.1514+37 654_1514+37 785 del; Chr11(GRCh37):g.2,720,964–2721,095del).


Molecular alterations of the IC2 in 11p15.5 are commonly associated with BWS, whereas only single cases with SRS features have been reported. In addition to gain-of-function variants of CDKN1C,14 they comprise duplications and deletions of several kilobases within or of the whole IC2 (for review).3–5 Microdeletions within the paternal IC2 allele have only been described in five families and affected the KCNQ1OT1 gene, the KCNQ1OT1:TSS-DMR/IC2 and at least parts of the coding sequence of the KCNQ1 gene (table 1 and figure 4). All variants should result in silencing of the paternally expressed KCNQ1OT1 gene and thereby should cause expression from the paternal copy of CDKN1C. In fact, decreased KCNQ1OT1 and increased CDKN1C expression have been proven in one of the reported cases.15 However, in two other cases with different sizes of deletions on the paternal allele, normal phenotypes and normal expression of the two genes have been documented.12 16 These observations are attributable to three CDKN1C enhancer sequences located within the KCNQ1 gene.13 Deletions of varying sizes and positions within this region thus have different impacts on CDKN1C expression and thereby cause phenotypes of variable severity (table 1).

Table 1

Overview of published cases carrying small deletions affecting the IC2 in 11p15.5 only.

Figure 4

Custom track illustrating the extent of the five currently known small deletions affecting the KCNQ1OT1:TSS-DMR/IC2. In three cases (marked by asterisks) the exact physical breakpoint positions were unknown.

The 132 bp deletion in the family described here is the smallest deletion in the IC2 reported so far and provides several insights into the function of the IC2 and the genes regulated by this imprinting centre.

  • According to its imprinting status as maternally methylated DMR (differentially methylated region), the deletion only causes clinical features in case the paternal allele is affected.

  • The deletion affects the paternal copy of the paternally expressed non-coding RNA (ncRNA) KCNQ1OT1 and therefore causes an overexpression of the cell-cycle inhibitor CDKN1C, which is normally silenced by the ncRNA on the paternal chromosome (figure 1). As a consequence, the patient is growth retarded. In contrast, her father is of normal growth as he carries the deletion on his maternal chromosome 11p15.5 on which KCNQ1OT1 is methylated and silenced and from which CDKN1C is expressed.

  • The growth retardation in our patient fits with the suggested enhancer model for the CDKN1C gene.4 13 The 132 bp deletion does not affect any of these enhancer motifs (figure 1), and therefore they all enhance the expression of CDKN1C from the paternal allele and thus promote the growth-inhibiting effect.

  • The methylation of the IC2 in the patient and her father is not generally altered, but the CpG within the deletion is lost, causing an altered hybridisation pattern of one MLPA probe. This finding is in contrast to the observation in a case of larger deletion (>60 kb), where the methylation of all MLPA probes is altered, in which either all CpGs are affected, or disrupted KCNQ1 transcripts cause an IC2 loss of methylation (LOM) (table 1).9–11

  • The normal ECG results confirm that the KCNQ1 transcript is not affected in our patient due to the intronic localisation of the 132 bp deletion.

In fact, we cannot finally exclude the coincidental occurrence of the growth retardation phenotype and the IC2 deletion in our patient without functional link. However, the identification of this small deletion in our growth retarded patient corroborates the suggestion that IC2 variants are involved in the aetiology of growth retardation and SRS features.4 In fact, these alterations are probably rare, as only single cases have been reported despite the broad application of MLPA assays targeting both the IC1 and the IC2 in the diagnostics of SRS and overlapping phenotypes. Nevertheless, testing of the IC2 should be included in the diagnostic work-up in growth retarded children with SRS features because it allows the detection of both rare molecular changes as well as unexpected findings17 to further enlighten the contribution of IC2 to growth and its disturbances.



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  • Contributors All authors have planned the study. TE prepared the manuscript and the other authors approved it. KE documented and evaluated the clinical data. FK and EL conducted the laboratory parts and performed the data interpretation. EK overviewed the study, and contributed to the clinical section and in the diagnosis of the family.

  • Funding The group is funded by the Deutsche Forschungsgemeinschaft (DFG, EG110/15-1).

  • Competing interests None declared.

  • Patient consent for publication Parental/guardian consent obtained.

  • Ethics approval The study was approved by the ethical committee of the Medical Faculty of the RWTH Aachen (EK159-08).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data sharing not applicable as no data sets generated and/or analysed for this study. All data relevant to the study are included in the article or uploaded as supplementary information. No data are available.