An 11p15 Imprinting Centre Region 2 Deletion in a Family with Beckwith Wiedemann Syndrome Provides Insights into Imprinting Control at CDKN1C

We report a three generation family with Beckwith Wiedemann syndrome (BWS) in whom we have identified a 330 kb deletion within the KCNQ1 locus, encompassing the 11p15.5 Imprinting Centre II (IC2). The deletion arose on the paternal chromosome in the first generation and was only associated with BWS when transmitted maternally to subsequent generations. The deletion on the maternal chromosome was associated with a lower median level of CDKN1C expression in the peripheral blood of affected individuals when compared to a cohort of unaffected controls (p<0.05), however was not significantly different to the expression levels in BWS cases with loss of methylation (LOM) within IC2 (p<0.78). Moreover the individual with a deletion on the paternal chromosome did not show evidence of elevated CDKN1C expression or features of Russell Silver syndrome. These observations support a model invoking the deletion of enhancer elements required for CDKN1C expression lying within or close to the imprinting centre and importantly extend and validate a single observation from an earlier study. Analysis of 94 cases with IC2 loss of methylation revealed that KCNQ1 deletion is a rare cause of loss of maternal methylation, occurring in only 3% of cases, or in 1.5% of BWS overall

Pedigree of the three generation family showing individual 11p haplotypes.

<p>Affected individuals are indicated by full shading, carriers by partial shading. Haplotypes are represented by the allele sizes for each marker in descending order (telomere to centromere), <i>D11S576, D11S922, TH, D11S4088, 244.14</i> and <i>HBB</i>. Inferred marker deletion is indicated by a dash.</p

GUSB copy number distribution in the BWS and control population.

<p>(a) GUSB expression values were obtained from a 50 ng equivalent of total RNA. Quantitative analysis of CDKN1C expression. (b) CDKN1C expression in the blood from 12 unaffected individuals (diamonds), five BWS cases with loss of methylation at IC2 and no deletion (squares), and individuals with deletion of the maternal IC2 (triangles). The Mann Whitney non-parametric U test was used to derive the P values shown. Statistically significant P values were obtained for the population differences in CDKN1C expression between unaffected controls and cases with loss of methylation and KCNQ1/IC2 deletion.</p

Q-PCR determination of copy number.

<p>Relative gene copy number analysis for amplicons across the KCNQ1 and flanking loci determined by Q-PCR. The heading KCNQ1OT1 refers to DNA within the KCNQ1OT1 region. The values shown are the ratio of the means of triplicate copy number determinations relative to the mean of the CFTR reference gene copy number. Deletion within the KCNQ1 locus is suggested by reduced copy number in the DNA from I-2, II-1, II-2 and III-1. Normal copy number is maintained in I-1.</p

Model of normal and disrupted imprinting regulation at the CDKN1C locus.

<p>Genomic distances were calculated from NCBI reference sequence NG_008935.1. (a) CDKN1C imprinting is maintained on the paternal chromosome by KCNQ1OT1 transcription leading to bidirectional gene silencing (indicated by spreading cloud) and formation of a chromatin insulator within the KVDMR (CTCF binding) that prevents a dominant distant CDKN1C enhancer from activating paternal CDKN1C expression. On the maternal chromosome, methylation within the KVDMR prevents chromatin insulator formation, KCNQ1OT1 is silent, and the distance enhancer can activate maternal CDKN1C expression. (b). In BWS cases with LOM within KvDMR, bidirectional gene silencing on both paternal and maternal chromosomes occurs, chromatin insulators exist on the maternal and paternal chromosomes and distant CDKN1C enhancers can no longer activate maternal CDKN1C expression. Mosaicism for the LOM epimutation allows some CDKN1C expression to occur. (c). Maternal deletion of 330 kb within the KCNQ1 locus removes the KvDMR, and the distant enhancer element, thereby reducing the maternal CDKN1C expression that is normally activated by distant enhancers. Enhancers located close to CDKN1C, not affected by deletion, may still be active and allow CDKN1C expression in some tissues. (d). Paternal deletion of 330 kb has a minimal effect on CDKN1C expression because even though the KvDMR is deleted on the paternal chromosome, the distant enhancer for CDKN1C is also deleted, neutralizing any potential for activation of paternal CDKN1C. Proximal CDKN1C enhancers unaffected by deletion may be active in some tissues. Maternal CDKN1C expression is unaffected.</p

Methylation sensitive MLPA.

<p>MS-MLPA analysis of the pedigree showing deletion from exon 2 to exon 15 within KCNQ1 in I-2, II-1, II-2 and III-1 (bolded), and maintenance of normal copy number in the remaining unaffected individuals. Normal methylation was maintained at the H19DMR (IC1) in all individuals and KvDMR methylation is abnormal in I-2, II-1, II-2 and III-1 (bolded). In I-2 the KvDMR methylation values are indicative of retention of the methylated IC2 on the maternal KCNQ1 allele and loss of the paternal unmethylated IC2, and in II-1, II-2 and III-3 these values are consistent with loss of the methylated IC2 on maternal KCNQ1 and retention of the paternal unmethylated IC2.</p

Southern blotting examining methylation at a Not1 site within KvDMR.

<p>The methylated maternal allele is at 4.2 kb and the unmethylated paternal allele is at 2.7 kb. Loss of the methylated maternal allele is shown for the affected individuals, II-1, II-2 and III-1. The methylated maternal allele is retained in I-2 and the unmethylated paternal allele is lost. Control DNA is represented by N and a positive control with loss of maternal allele methylation is in the adjacent lane.</p