Laboratory Issues

While UPD is readily detected by DNA marker analysis, both parents may not be available for testing. In such cases, analysis of one parent and the child still may be successful for identification of UPD for a particular chromosome. For example, if a mother-child pair is analyzed, the absence of maternal alleles suggests paternal UPD for a specific chromosome, while the converse situation is true with a father-

child pair. Markers from other chromosomes may be used to confirm parentage.

In addition to UPD, absence of DNA marker alleles inherited from a parent may arise as the result of sub-microscopic chromosome deletion. Fluorescent in situ hybridization (FISH) analysis is used to confirm microscopic deletions in cases where multiple DNA markers from the same chromosome indicate an absence of marker contribution from one parent in one chromosomal segment yet the presence of normal biparental inheritance in another segment. Routine chromosome analysis (kary-otyping) should be used to rule out large deletions as part of a full patient study.

Commercial kits are not available for DNA marker analysis of all chromosomes. However, oligonucleotide primers for amplification of marker loci easily can be synthesized and obtained from numerous companies. Depending on the disorder, cell lines and DNA from patients with specific UPD-associated disorders may be available from the Coriell Cell Repositories ( Although proficiency testing for Prader-Willi and Angelman syndromes is available, to date no proficiency test is available specifically to assess proficiency in UPD testing.


Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by a variety of genetic abnormalities involving the proximal part of the long arm of chromosome 15 (q11-q13). Within a 2 megabase (Mb) domain, gene expression depends on the parent of origin resulting from imprinting. Several genes defined above in the region (SNRPN [small nuclear ribonu-clear protein N], ZNF127, IPW, PARI, PAR5, PW71, NECDIN) are expressed exclusively from the paternal chromosome in all tissues studied, whereas another gene, UBE3A (ubiquitin-protein ligase E3A), is expressed only from the maternal chromosome in brain but has biparental expression in other tissues. Evidence suggests that PWS is caused by loss of expression of one or more of the C/D box small nucleolar RNAs (snoRNAs) encoded within the SNRPN locus.11 On the other hand, AS results from loss of expression of UBE3A, which is involved in the ubiquitina-tion pathway targeting certain proteins for degradation.12 Imprinting of genes in this domain is coordinately controlled by a bipartite imprinting center (IC) overlapping the SNRPN promoter and extending 35kb upstream. Flanking the imprinted region and approximately 4Mb apart are several low-copy repeats derived from an ancestral HERC2 gene and other sequences that predispose the region to chromosomal rearrangement by unequal crossing over.

All the genetic mechanisms identified in PWS interfere with the expression of paternally expressed genes in the 15q11-q13 domain.13 Approximately 70% of PWS patients have a 4Mb deletion of the paternal chromosome 15q11-13, which occurs sporadically through unequal crossing over. Maternal UPD for chromosome 15 accounts for about 20% of PWS and is usually the consequence of abnormal chromosome segregation during meiosis. In 1% to 5% of cases, microdeletions (6 to 200 kb) that include the IC or epigenetic changes in the IC occur and are inherited in an autosomal dominant manner. Such defects result in both maternal and paternal copies of genes in the imprinted domain having a maternal imprint and therefore lacking expression of the paternally inherited genes. Finally, about 1% of patients have translocations or other structural abnormality involving chromosome 15, which result in deletion or maternal UPD of the 15q11-q13 region.

Four different genetic mechanisms have been identified in 85% to 90% of AS patients, and all cause loss of UBE3A expression.14 A 4Mb deletion of the maternal chromosome 15q11-q13 region occurs sporadically in about 65% to 70% of cases due to unequal crossing over, as occurs in PWS. Paternal UPD has been detected in about 3% to 5% of patients and is also due to abnormal segregation of chromosome 15 in meiosis. Approximately 7% to 9% of AS patients have an imprinting defect that results from microdeletions of the IC or epigenetic changes in the IC. Such defects cause lack of expression of the maternally inherited UBE3A gene in brain because both maternal and paternal copies have a paternal imprint. Point mutations in the UBE3A gene (mostly truncating mutations) are found in 4% to 11% of AS patients.15 Approximately 10% to 15% of patients with a clinical diagnosis of AS have no identifiable chromosomal or molecular abnormality. It is thought that these patients have some undetected abnormality that affects the UBE3A gene or have a mutation in another gene in the ubiquitination pathway.

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