1. 11p15 familial duplication causing Silver-Russell syndrome in the daughter and Beckwith-Wiedemann syndrome in the mother R. Teek 1, T. Kahre 1,2, K. Muru 1,2, O. Zhilina 1,3, P. Mee 4, K. Õunap 1,2. 1 Department of Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia 2 Department of Pediatrics, University of Tartu, Tartu, Estonia 3 Department of Biotechnology, Institute of Cell and Molecular Biology, University of Tartu, Tartu, Estonia 4 United Laboratories, Tartu University Hospital, Tartu, Estonia Case report: Phenotypes of the child and her mother Introduction:The pedigree MS-MLPA Conclusion SRS and BWS have been linked with a variety of epigenetic and genetic defects affecting a cluster of imprinted genes at chromosome 11p15.5. More than 50% BWS patients have aber- rant methylation patterns in 11p15. In SRS patients hypomethylation was found in up to 63%; 11p15 duplication of maternal chromosome was found in only 1-2%. To date only one report is published about 11p15 duplication resulted in SRS and BWS phenotype in single family. Chromosomal microarray analysis (CMA) Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous disorder characterized by severe intrauterine and postnatal growth retardation. Beckwith-Wiedemann syndrome (BWS) is defined by overgrowth, hemihyperplasia and an increased risk of childhood tumors. It is known that SRS and BWS are both resulted by methylation defects in chromosomal region 11p15. Most of cases SRS and BWS are sporadic but some familial cases are also reported. We report here a familial case, where daughter has SRS and her mother BWS caused by duplication in 11p15 chromosomal region. Childs’ SRS diagnostic clinical features: Mothers’ BWS diagnostic clinical features: Birth weight <10 percentile Birth lenght <10 percentile Relative macrocephaly at birth Short stature <3 percentile Head circumference 3-97 percentile Normal cognitive development Asymmetry of body Triangular face Prominent forehead Small chin Clinodactyly Macrosomia Anterior linear ear lobe creases Positive family anamnesis for BWS Characteristic face Figure 2. The child at newborn age. Figure 3. The mother and the child. We performed CMA for the child and found ~1.3-Mb size duplication in chromosomal region 11p.15.5. CMA of her parents revealed that the duplication is inherited from her mother. Figure 4-5. CMA results of child (A9553) and her mother (A9898) Figure 1. Epigenetic regulation of the two imprinting control regions (ICRs) in 11p15 and illustration of the hyper- /hypomethylation of the ICR1 observed in BWS and SRS. In the ICR1, hypermethylation of the H19 promotor and loss of imprinting of IGF2 are detectable in 2–7% of BWS patients, whereas loss of methylation in the same region is a frequent finding in SRS. The majority of BWS shows hypomethylation in the ICR2 or mutations in the ICR2-regulated CDKN1C gene Eggermann et al., 2008 This work was supported by grants GARLA 8175 and TARLA 2695. Acknowledgements Figure 6. The pedigree for reported family Proband, SRS phenotype BWS phenotype 11p15.5 duplication in CMA ab cd Figure 7. MS-MLPA final probe ratios in a) copy number variation analysis (CNV) in a normal reference subject (normal ratio between 0.75 and 1.3); b) CNV analysis of the child with 11p15.5 duplication involving region 11-001973915 to 11-002863231 (probes average ratio 1.323) c) methylation status analysis of normal reference: in IC1 (mean of 4 MS probe ratios 0,55; ±2SD 0,53-0,57) and in IC2 (mean of 4 MS probe ratios 0.60±2SD 0.55-0.64); d) methylation status analysis of the child with 11p15.5 duplication: hypomethylation of IC1 (mean of 4 probe ratios 0.39) and hypermethylation of IC2 (mean of 4 MS probe ratios 0,69) Blue rectangle - range of final probe ratios in reference samples (N=5); red rectangle - final probe ratio of investigated subject ±2SD Methods: The MS-MLPA analysis was made with probe mixture ME030-B2BWS/SRS by manufacturers protocols (MRC-Holland, Amsterdam, NL). This mix contains 45 probes, 27 of which are specific for the BWS/SRS region in 11p15 including 13 methylation specific probes containing HhaI recognition site. In particular, 5 methylation-specific (MS) probes locate inside IC1 (H19DMR), 3 MS probes in IGF2 (DMR0), 6 MS probes hybridize to the KCNQ1 including IC2 region (KvDMR). PCR products were separated using ABI 3130XL Genetic Analyzer and data analyzed by using Coffalyser programm (MRC- Holland). All samples underwent intrasample normalisation, obtained Normalization Constant is used for sample to reference comparison in each probe. To analyze methylation status of MS-MLPA probes Normalization Constant of each digested MS-MLPA probe should be divided by the Normalisation Constant obtained from each corresponding undigested MS-MLPA probe. Aberrant methylation can be identified by comparing the methylation status of MS-MLPA probes in the examinated sample to that obtained on reference samples. References The CMA analysis was performed with the Illumina HumanCytoSNP-12 version 1.0 BeadChip (Illumina Inc., San Diego, CA, USA5) including 301,232 markers that cover the whole genome with median spacing of 6.2 kb. Cardarelli L, Sparago A, De Crescenzo A, Nalesso E, Zavan B, Cubellis MV, Selicorni A, Cavicchioli P, Pozzan GB, Petrella M, Riccio A. Silver- Russell syndrome and Beckwith-Wiedemann syndrome phenotypes associated with 11p duplication in a single family. Pediatr Dev Pathol. 2010 Jul- Aug;13(4):326-30. Eggermann T. Russell-Silver syndrome. Am J Med Genet C Semin Med Genet. 2010 Aug 15;154C(3):355-64. Review. Eggermann T, Begemann M, Binder G, Spengler S. Silver-Russell syndrome: genetic basis and molecular genetic testing. Orphanet J Rare Dis. 2010 Jun 23;5:19. Review. Eggermann T, Eggermann K, Schönherr N. Growth retardation versus overgrowth: Silver-Russell syndrome is genetically opposite to Beckwith- Wiedemann syndrome.Trends Genet. 2008 Apr;24(4):195-204. Epub 2008 Mar 7. Review. Weksberg R, Shuman C, Beckwith JB. Beckwith-Wiedemann syndrome. Eur J Hum Genet. 2010 Jan;18(1):8-14. Epub. Review.