A Beckwith-Wiedemann syndrome case with de novo 24 Mb duplication of chromosome 11p15.5-14.3

Background: Molecular genetic testing for the 11p15-associated imprinting disorder Beckwith-Wiedemann syndrome(BWS) is challenging because of the molecular heterogeneity and complexity of the affected imprinted regions. An accurate diagnosis of BWS requires a complete molecular method to analyze epigenetic changes. Case presentation: We reported a Chinese case with BWS detected by SNP array analysis and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The genetic analysis showed a de nove duplication of 24 Mb at 11p15.5-14.3 is much longer than ever reported. MS-MLPA showed copy number changes with a peak height ratio value of 1.5(three copies) at 11p15. The duplication of paternal origin with increase of methylation index of 0.68 at H19 and decreased methylation index of 0.37 at KCNQ1OT1 . Conclusion: Combined chromosome microarray analysis and methylation profiling provided reliable diagnosis for this paternally derived duplication of BWS. The phenotype associated with 11p15 duplications depends on the size, genetic content, parental inheritance and imprinting status. Identification of these rare duplications is crucial for genetic counselling.

We investigated a patient with clinical features of BWS. Genetic analyses revealed that the patient harbored pUPD and copy number variation. This is a rare reported case of a patient with 24Mb duplication， which is much longer than ever reported.

Patient report
The female infant was the first-born baby to non-consanguineous, healthy, Chinese parents after 36 weeks and 6 days of gestation in Jiaxing maternal and Child Health Hospital. Her birth weight was 3790 g, and she exhibit edcolumnar head, collapse of nasal bridge, wide distance of eyes, right hand through the palm, mild tricuspid regurgitation(Opposite velocity: 2.88M/S, PG:33.2mmHg), patent ductus arteriosus, the right ventricle is markedly enlarged (Fig.1a). Bilateral ventricle dilation(

DNA isolation
Sample was collected and genomic DNA was extracted from the peripheral blood of the patient. Isolation and purification of the genomic DNA were performed using a Qiagen DNeasy Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany).

SNP array analysis
Single nucleotide polymorphism (SNP) array analysis was performed with CytoScan 750K/HD array(Carlsbad, CA, USA) according to the manufacturer's protocol. The microarray was used to investigate the CNVs and absence of heterozygosity(AOH) events.
The locations of the CNVs and the UPD events were determined based on a human genome assembly from February 2009 (GRCH37/h19).
OMIM genes and Ref-Seq genes were used to evaluate the CNVs identified in this study. The criteria used for interpreting whether a CNV was pathogenicor benign were according to the guidelines recommended by the American College of Medical Genetics (ACMG) [9].

MS-MLPA Analysis
MS-MLPA analysis was used to validate the results observed with CMA. MS-MLPA for 11p15.5 showed copy number changes with a peak height ratio value of 1.5(three copies) at 11p15 (Fig.1f) in comparison with a ratio value of 1(two copies) from a normal control. MS-MLPA indicated methylation index of 0.68 at IC1 and methylation index of 0.37 at IC2 (Fig.1g). We indentified this patient with duplication of the BWS critical region.

Discussion
In this study, we report a Chinese BWS case with de novo paternally Although our patient's clinical phenotype fits well to this description, and the results from SNP array and MS-MLPA analysis fulfilled the diagnostic criteria for BWS, the 24 Mb duplication at 11p15.5-14.3 is much longer than ever reported [15][16]. Qin Wang reported two Chinese cases with BWS, One case was a de novo paternal origin duplication spanning 896Kb at 11p15.5. Case 2 was referred at 2-month old and the genetic analysis showed a de novo 228.8Kb deletion at 11p15.5 telomeric end and a de novo duplication of 2.5 Mb at 11p15. 5-15.4. Both duplications are of paternal origin with gain of methylation at the imprinting center 1 [14]. In our case, the de novo duplication of 24 Mb is much longer and involving more genes.
We consider that most of the symptoms in our patient is caused or modulated by the duplication of these genes, including the OMIM genes H19, IGF2, TH, KCNQ1, STIMI and so on, involving 210 OMIM genes.

H19(103280) plays a key role in the development of
Beckwith-Wiedemann syndrome, Silver-Russell syndrome and it had been hypothesized that loss of H19 expression may be involved in Wilms tumorigenesis [17]. H19 is a developpmentally regulated gene with putative tumor suppressor activity. IGF2 (147470) is a protein hormone involved in the regulation of cell proliferation, growth, migration, differentiation, and survival. It has been found that aberrant processing of pro-IGF2 by PCSK4 may be a cause of intrauterine growth restriction, a leading cause of perinatal mortality [18]. The expression of the IGF2 and H19 genes is imprinted. Although these neighboring genes share an enhancer, H19 is expressed only from the maternal allele, and IGF2 only from the paternally inherited allele. The region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. The KCNQ1OT1(604115) gene was expressed preferentially from the paternal allele [19], while KCNQ1 transcription is silent. In most patients with BWS, KCNQ1OT1 is abnormally expressed from both the paternal and maternal alleles. 21 of 36 (58%) BWS patients showed loss of maternal allele-specific methylation of a CpG island upstream of KCNQ1OT1. The authors determined that LOI of KCNQ1OT1 is the most common genetic alteration in BWS [20].
Chang reported a patient with a loss of heterozygosity in the region of chromosome 11p14.3 to 11p15.5. This region is similar to the case in this article. The results suggest that paternal uniparental isodisomy and mosai cism [21].
In this case, because the increase of paternal gene's copy number, the H19 methylation index of 0.68 at IC1 is increased in comparison with normal control methylation index of 0.54 at IC1. And the KCNQ1OT1 methylation index of 0.37 at IC2 is decreased in comparison with normal control methylation index of 0.57 at IC2. A recent study in a serial of over 400 BWS cases also indicated that copy number changes in the 11p15.5 region contributed significantly to the etiology of the BWS [22].
For this patient, physical examination should be performed rotinely for potential intellectual disability and the possible clinical effect involving the deleted genes. The phenotype associated with 11p15 duplications depends on the size, genetic content, parental inheritance and imprinting status. Identification of these rare duplications is crucial for genetic counselling. Furthermore, SNP arrays can be helpful in clarifying the molecular diagnosis in patient with BWS, especially to discriminate between pUPD and duplications [23].

Funding
This work was funded by support from Zhejiang Provincial Natural

Conflict of interest
The authors have no competing financial interests to declare.