Available Assays

Clinical molecular testing for PWS and AS includes the molecular assessment of the parent-specific imprint within the 15q11-q13 region. Methylation,which is involved in the process of genomic imprinting, has been demonstrated for several of the genes in the imprinted domain, including

SNRPN. Standard molecular techniques for the methyla-tion analysis at the CpG island in exon a of the SNRPN gene include (1) double-digest Southern blot analysis using a methylation-sensitive enzyme such as Not I along with a methylation-insensitive enzyme such as Xba I,16 (2) PCR following either Not I or McrBC digestion with primers for the SNRPN promoter, and (3) methylation-specific PCR based on modifying DNA with bisulfite, which converts all unmethylated cytosines to uracils, followed by amplification using primers specific for the unmethylated and methylated alleles.17 The assessment of SNRPN expression by reverse transcription-PCR (RT-PCR) also may be used for the diagnosis of PWS.

For the purpose of genetic counseling, once the diagnosis of PWS or AS is established by abnormal methylation or SNRPN expression testing, further tests should be performed to determine the genetic mechanism responsible for the disorder. FISH of metaphase chromosomes using the probes SNRPN for PWS and either SNRPN or D15S10 for AS will detect the 4Mb deletion in the majority of patients. For patients without a deletion, DNA marker analysis should be used to detect UPD. Using specimens from both parents and the affected child. Mutations of the IC account for the remaining PWS patients and some of the AS patients, and referral may be made to a research laboratory for further investigation. Patients with an AS phe-notype but normal methylation should be assisted to have testing by a clinical laboratory offering mutation analysis of UBE3A.

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