Chiesa et al. (2012). The KCNQ1OT1 imprinting control region and non-coding DNA: new properties derived from the study of Beckwith-Wiedemann syndrome and Silver-Russell syndrome cases. Hum Mol Genet. 21(1): 10-25.

Introduction

• Imprinting control regions (ICRs) are cis-acting regulatory elements of the imprinted loci
• Chromosome 11p15.5 has a large cluster of imprinted genes
• Divided into 2 separate domains that each have their own ICRs
• ICR1 is telomeric, ICR2 is centromeric, have different mechanisms of action
• ICR2 is the promoter of KCNQ1OT1 gene (non-coding, imprinted)
  • KCNQ1OT1 is antisense, contained within the protein-coding KCNQ1 gene
  • Non-coding KCNQ1OT1 transcript silences imprinted genes of the centromeric domain on the paternal chromosome
  • Methylation of ICR2 on maternal chromosome, not transcribed and imprinted genes are expressed
• Changes in methylation patterns on the imprinting control regions (ICRs)
• Loss of methylation on the maternal allele of ICR2 results in BWS
  • Most common defect in BWS
  • Leads to bi-allelic activation of KCNQ1OT1 and silencing of the imprinted genes
  • This includes the cell growth inhibitor CDKN1C
• Inverse defects in DNA methylation at ICR1 causes BWS or SRS
  • With associated changes in the expression of IGF2/H19 transcripts
• Mutation of the cell growth inhibitor CDKN1C (5% of BWS)
• Loss-of-function mutation in a trans-acting factor demonstrated in familial case of BWS
• Uniparental disomy frequently observed in BWS
• Chromosomal abnormalities – rarer, usually involve paternal duplications, maternal deletions or balanced maternal translocations in BWS
  • Maternal duplication usually observed in SRS
• 2 cases in this paper:
  • 2 Mb long inverted duplication of entire imprinted gene cluster à SRS phenotype
  • 160 kb duplication including ICR2 and 5’ 20 kb of KCNQ1OT1 co-segregating with BWS phenotype in 3 generations
    • Expression of truncated KCNQ1OT1 transcript, silencing of CDKN1C results

Results

1. SRS family

• SRS patient born in family with no signs of SRS
  • Intra-uterine growth restriction, low birth weight and length
  • Height and weight below the 3^rd centile
• Slight ICR1 hypomethyation, ICR2 hypermethylation observed in patient
  • Parents were both normal at both loci (50%)
• Used microsatellite and SNP analysis to find de novo maternal duplication in the patient
• 2 Mb duplication encompassing the entire imprinted gene cluster, only in cis (confirmed via FISH)
• Hypermethylation of CpGs throughout SRS patient 1, consistent with duplication of methylated maternal allele
  • Shows duplicated chromosome acquired imprinted methylation of ICR2

2. BWS family

• Female patient born to unrelated parents
  • Mother born with elevated birth weight, father normal
  • Maternal uncle and aunt also had elevated birth weight
  • Umbilical hernia also runs in the family on the maternal side
• MS-MLPA – hypomethylation at ICR2 and normal methylation at ICR1 in III-6, II-4, II-3, I-4
  • Normal methylation in ICR2 and ICR2 at II-5, III-5
  • Increased copy number of KCNQ1 exons 12-15 and ICR2 in all family members with hypomethylation
  • Maternal transmission of 11p15.5 duplication from I-4 to II-3 and II-4, from II-4 to III-6
• Identified in cis duplication of a portion of the KCNQ1 gene (exon 12-15), ICR2 and most 5’ 20 kb of KCNQ1OT1
  • Excluded mutations of CDKN1C gene in patient 1 via exon sequencing
• Extensive hypomethylation at ICR2 in all individuals with the 160 kb duplication
  • Loss of imprinted methylation of maternal allele evident in BWS patients 1 and 2
  • 160 kb duplication leads to imprinting alteration and BWS phenotype only with maternal transmission
• By cloning the patient cells, determined that hypomethylation was likely due to lack of methylation of one of the two ICR2 copies present on the maternal chromosome
• Found KCNQ1OT1 gene expressed on the maternal chromosome
  • Bi-allelic expression of KCNQ1OT1 in duplication region but normal in non-duplicated region
• Level of CDKN1C RNA was lower in BWS patient 2 vs. age-matched controls
  • Indicates silencing of CDKN1C by expressed KCNQ1OT1
  • Confirmed that KCNQ1OT1 interacts with chromatin
  • Interaction exerted at least partially by 5’ 20 kb sequence
  • The silencing of CDKN1C likely occurs via interaction with chromatin