Interpretation of Test Results

If the methylation pattern or methylation-specific amplification is characteristic of only maternal inheritance, then the diagnosis of PWS is confirmed. If the methylation pattern or methylation-specific amplification is characteristic of only paternal inheritance,then the diagnosis of AS is confirmed (Figure 6-3). Methylation assays detect all cases of PWS and AS caused by deletions, UPD, and IC defects; however, such assays will not detect the rare small deletions not involving the SNRPN locus or UBE3A mutations. While the methylation assays cannot differentiate among the various mechanisms producing PWS and AS, 99% of PWS patients and approximately 78% of AS patients will be detected. Approximately 50% of the remaining patients with an AS phenotype have mutations in UBE3A, which are identified by single strand conformation polymorphism (SSCP) analysis and sequencing.

Two different approaches for the laboratory diagnosis of PWS and AS may be used.18 The first is to start with an analysis of parent-specific methylation of SNRPN. A pattern consistent with biparental inheritance rules out PWS and most cases of AS. If the diagnosis of PWS or AS is confirmed by methylation analysis, FISH and DNA marker analysis should be performed to determine whether the cause is a deletion, a UPD, or an IC defect. The second approach takes into account that deletions are the

Figure 6-3. Southern blot analysis of PWS and AS. The methylated 4.2 kb (maternal) and the unmethylated 0.9 kb (paternal) fragments generated using Xba I and Not I are detected by the DNA probe SNRPN. Normal individuals exhibit a biparental inheritance pattern (lanes 1 and 4), whereas patients with PWS show a pattern of only maternal inheritance (lane 2) and patients with AS show a pattern of only paternal inheritance (lane 3).

Figure 6-3. Southern blot analysis of PWS and AS. The methylated 4.2 kb (maternal) and the unmethylated 0.9 kb (paternal) fragments generated using Xba I and Not I are detected by the DNA probe SNRPN. Normal individuals exhibit a biparental inheritance pattern (lanes 1 and 4), whereas patients with PWS show a pattern of only maternal inheritance (lane 2) and patients with AS show a pattern of only paternal inheritance (lane 3).

most common cause of PWS and AS. It may be preferable to perform FISH analysis first if the patient is an older child or adult with classic features of PWS or AS. Methylation analysis is appropriate if a deletion is not detected. In the case of abnormal methylation, UPD studies using DNA markers should be performed to determine whether UPD is the cause. For either approach,if UPD is not present,then referral to a clinical laboratory for mutation analysis of UBE3A should be considered for patients with an AS phe-notype. For patients suspected to have PWS, referral to a research laboratory for further molecular investigation of an IC defect should be considered.

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