EP311: Diagnostic testing for Beckwith-Wiedemann syndrome/Russell-Silver syndrome: The GGC experience

Abstract

Beckwith-Wiedemann syndrome (BWS) and Russell-Silver syndrome (RSS) are congenital imprinting disorders commonly caused by methylation defects in the same region of chromosome 11p15. BWS is an overgrowth syndrome that includes clinical features such as macroglossia, organomegaly, umbilical hernia, hypoglycemia, and an increased risk of childhood tumors. RSS is characterized by intrauterine and postnatal growth retardation, relative macrocephaly, fifth finger clinodactyly, triangular facies, and body asymmetry. BWS and RSS are caused by reciprocal defects of the Imprinting Control Regions (ICR) at chromosome 11p15. BWS is caused by microduplications/microdeletions of 11p15, gain of maternal methylation at ICR1, loss of maternal methylation at ICR2, paternal UPD11, and maternally inherited pathogenic variants in the CDKN1C gene. RSS is mainly caused by maternally inherited duplications of 11p15, loss of paternal methylation at ICR1, and maternal UPD7 in approximately 10% of cases. The molecular diagnostic laboratory at the Greenwood Genetic Center (GGC) offers molecular testing for BWS and RSS syndromes. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) is the first tier of testing. This test can simultaneously detect the methylation and copy number status of the differentially methylated regions, ICR1 and ICR2, at 11p15. Abnormal MS-MLPA results are confirmed by Pyrosequencing, an orthogonal method that detects the quantitative percent of methylation at ICR1 and ICR2, which also enables the detection of mosaicism; an in-house mosaic calculator has been developed for this analysis. Cases negative for RSS by MS-MLPA can be reflexed to UPD7 testing. Sanger sequencing of the CDKN1C gene and UPD7 testing are available at GGC as reflex tests for cases with negative BWS MLPA or RSS MLPA results, respectively. A total of 867 cases have been included in this study. For BWS, the diagnostic yield by MS-MLPA is 21.6% (117/541), where loss of methylation at ICR2 (87 cases), gain of methylation at ICR1 (3 cases), and copy number change (5 cases) were detected. Among these BWS MLPA positive cases, 18.8% (22/117) were due to paternal UPD11, which included six cases that resulted from mosaicism. Approximately, 23.3% (99/424) of BWS MLPA-negative cases had CDKN1C sequencing, and variants of uncertain clinical significance (VUS) were identified in four cases. For RSS, the diagnostic yield by MS-MLPA is 12.5% (41/326) of which three cases had a mosaic loss of methylation at ICR1 and two cases had copy number changes. 21.7% (62/285) of RSS MLPA-negative cases were reflexed to UPD7 testing, which increased the diagnostic yield to 14% (46/326). It is of interest to note that eight cases submitted for BWS testing were found to be abnormal for RSS syndrome. The clinical finding of hemihypertrophy in these cases prompted testing for BWS; this raises the question of whether hemihypoplasia was being interpreted as hemihypertrophy in these cases since loss of methylation at ICR1 has been observed in isolated cases of hemihypoplasia. Similarly, two cases that were submitted for RSS testing was found to be abnormal for BWS. This finding indicates that hypomethylation of both ICR1 and ICR2 has been identified in patients with RSS, and it is thought that epigenetic mosaicism may exist between relevant disease tissues, which can result in clinical expression of either RSS or BWS. In summary, by utilizing various methods of testing for BWS/RSS (MS-MLPA, Pyrosequencing, Sanger sequencing, and UPD testing), the collective diagnostic yield is approximately 19%. Pyrosequencing can confirm an abnormal finding by MS-MLPA and is a quantitative method that can be utilized for detecting percent mosaicism. Finally, for RSS negative cases, reflex testing for UPD7 can increase the diagnostic yield.