Laboratory Testing for Prader-Willi Syndrome 4

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4

Laboratory Testing for Prader-Willi Syndrome

Kristin G. Monaghan and Daniel L. Van Dyke

In 1981, Ledbetter and co-workers⁴⁵ described a cytogenetically visible deletion in proximal 15q in four patients with Prader-Willi syndrome (PWS). Proximal 15q was targeted for analysis because several previ- ously published Prader-Willi cases exhibited an isochromosome 15q or a translocation with proximal 15q breakpoints.^12,34 Since the ﬁ rst report of the cytogenetic deletion of proximal 15q in PWS, several cytogenetic and molecular genetic techniques have been used to further character- ize the chromosome 15q11–q13 region and for laboratory diagnostic purposes in a range of PWS subjects having a variety of abnormal chromosome ﬁ ndings.

Using several chromosome staining methods to evaluate chromosome 15 short arm variants, Butler and Palmer¹⁶ showed that the PWS deletion preferentially involves the paternally inherited chromosome 15. Butler¹³ subsequently reported that short arm or C-band variants appeared to be more common in chromosome 15s with a deletion than in normal 15s.

Nicholls, Butler, and co-workers⁵⁶ studied the parental origin of the chromosome 15 pair in two PWS patients who did not exhibit a 15q deletion. They were the ﬁ rst to describe uniparental disomy (UPD) in PWS and concluded that genetic imprinting in this region must play a causal role in both PWS and Angelman syndrome (AS). One of their PWS patients carried a familial robertsonian 13;15 translocation. What they could not recognize at the time was the causal relationship between robertsonian translocations and UPD.³

Aside from these important early studies, a large variety of balanced and unbalanced chromosome 15 rearrangements have been described in PWS patients.^12,17 Most of the chromosome 15 rearrangements associated with PWS have resulted in deletion of proximal 15q, and in many of these cases deletion of the paternally inherited chromosome 15 was demonstrated. Some unusual chromosomal causes of PWS that have been described include intrachromosomal triplication⁷¹ and an unbalanced reciprocal translocation resulting in maternal disomy for proximal 15q.⁵⁸ In contrast to the deletion cases, PWS and AS patients who 74

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carry an additional dicentric chromosome [+dic(15)(q11-q13)] also have UPD, which accounts for their phenotype.⁶¹ Patients with a +dic(15)(q11–

q13) without UPD have a different phenotype.^51,59

Hulten et al.³⁷ described a family segregating a translocation involving chromosome 15 and 22 [t(15;22)(q13;q11)] in affected PWS and AS children. The affected children exhibited the same 15q deletion and 22q duplication, except that the PWS children inherited the translocation from their carrier father, and the AS child inherited it from his carrier mother.

A landmark report by Dittrich et al.²⁶ described the abnormal DNA methylation pattern in PWS and AS. They digested patient DNA with the methylation-sensitive HindIII/HpaII restriction enzymes and performed Southern blot analysis with the PW71 (D15S63) probe. Normal subjects exhibited a 6.0 kb and 4.4 kb band, whereas PWS subjects exhibited only the maternal 6.0 kb band and AS subjects exhibited only the paternal 4.4 kb band.

In 1994, Lerer et al.⁴⁷ reported their use of probe PW71 to detect parent-of-origin-speciﬁ c differences in DNA methylation at the D15S63 locus in PWS patients with 15q deletions or UPD. Using similar methods, Butler¹⁴ evaluated a group of 27 suspected PWS patients.

Thirteen had a deletion by G-banding and FISH. One patient had a balanced 15;19 translocation, four had UPD, and two had no parental studies but exhibited abnormal methylation, so probably had UPD.

Two patients with normal karyotypes exhibited a normal methylation pattern and so remained unexplained. Testing ruled out PWS in the remaining fi ve patients. The patient with the t(15;19) exhibited a normal methylation pattern, but the FISH results were consistent with a chromosome 15 break within the PWS critical region.⁶⁷ This important patient appears to represent the fi rst with a defi nite PWS diagnosis and a normal methylation pattern. A few similar PWS patients have been described with a balanced translocation and a normal DNA methylation pattern.¹⁷ In the cases that have been evaluated, the translocation breakpoint was within the SNRPN gene or between the SNRPN and IPW genes.22,43,63,67 Each of these translocations represented a new mutation in the patient.

Several reports have described multiplex PWS families.^50,52 In each case, the methylation pattern of affected PWS individuals and carrier family members, such as fathers, was abnormal and consistent with a diagnosis of PWS. The affected family members inherited an atypically small deletion—as small as 7.5 kb—from their father, and the unaffected carriers inherited the deletion from their mother. Such deletions are important to identify because there is a 50% risk of PWS in children of carrier males. For example, the family described by McEntagart et al.⁵⁰ exhibited the usual PWS methylation pattern. Microsatellite analysis was employed to distinguish between UPD and a deletion. Four microsatellite markers within the PWS critical region demonstrated normal biparental inheritance, but two other markers exhibited only a maternal allele. Additional family studies conﬁ rmed an inherited dele- tion of the markers D15S128 and D15S63. The unaffected father and paternal grandmother of the proband exhibited a typical AS methyla-

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tion pattern, or having no maternal contribution, and this inheritance pattern has been observed in other families (cf. Buiting et al.⁸). In these families, detailed genetic services and counseling are recommended.

Ohta et al.⁵⁷ described sporadic PWS patients with abnormal methylation but without evidence of UPD, a deletion involving the PWS critical region, or a deletion of the imprinting center. In contrast to the imprinting center deletion families, these cases appear to have no signifi cant recurrence risk. Buiting et al.⁹ summarized their observations of 51 PWS and 85 AS patients who exhibited an imprinting defect (abnormal methylation pattern) but who did not have evidence of the usual deletion or UPD. Seven of the 51 PWS patients were shown to have a deletion involving the imprinting center, and in fi ve of these subjects the father carried the same deletion on the chromosome 15 that he inherited from his mother. Three of the PWS patients with an imprinting center deletion had a positive family history. In contrast, none of the remaining 44 patients had an affected relative. Sequence analysis of the PWS-SRO (PWS-shortest region of deletion overlap) for 32 of the 44 nondeletion patients revealed no clinically signifi cant mutations—only benign, single nucleotide polymorphisms. Parental and grandparental origin studies were most consistent with PWS having been caused by a rare and sporadic failure during spermatogenesis to erase the maternal imprint from the PWS/AS imprinting center.

FISH Probes for Prader-Willi Syndrome Diagnosis

Several FISH probe kits are commercially available that employ a probe encompassing the SNRPN and IC region, and a control probe localized to distal 15q. Metaphase FISH analysis is expected to reveal a normal pattern with a SNRPN/IC signal and a control signal on each chromo- some 15, or a deletion pattern with a control signal on each chromo- some 15 but only one SNRPN/IC signal. A normal FISH pattern does not exclude UPD or a microdeletion involving the IC only. Some but not all chromosome 15 rearrangements are detectable using FISH (e.g., isochromosome 15q or extra dicentric 15).

Cytocell (www.cytocell.com) manufactures the Aquarius Prader- Willi/Angelman Region Probe with Control Probe (15qter). This probe kit targets SNRPN and the imprinting center. Vysis (www.vysis.com) manufactures four PWS/AS region probe kits. Among them, the LSI Prader-Willi/Angelman Region Probe (SNRPN) targets SNRPN and includes control probes for the centromere region and 15q22 (PML).

Molecular Analysis for Prader-Willi Syndrome

Molecular genetic testing for Prader-Willi syndrome can be divided into four categories: methylation analysis by Southern blot or polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR) to detect SNRPN expression, microsatellite analysis to detect uniparental disomy, and specialized studies using various molecular techniques for

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the identiﬁ cation of imprinting defects. Each method has advantages and disadvantages compared with other methods (Table 4.1). For some patients, a combination of several methods may be needed to establish a diagnosis and determine the etiology (Figure 4.1). Genetic testing and genetic counseling for Prader-Willi syndrome are important not only to conﬁ rm the diagnosis but also to determine and discuss the recurrence risk of PWS with other family members. The recurrence risk is less than 1% if the proband has a large deletion, uniparental disomy, or a nondeletion imprinting defect. Imprinting defects that are due to a microdeletion in the imprinting center (IC) are associated with a 50%

risk of PWS in the siblings of a proband. Familial PWS deletions are found on the paternal chromosome of the proband and on the maternal chromosome 15 of the phenotypically normal father.

Molecular genetic tests currently used for the diagnosis of PWS are based on the difference in the methylation status of the maternal and paternal chromosome 15s. The difference occurs within genomic areas consisting of a high proportion of the dinucleotide CpG, referred to as CpG islands. CpG islands are located throughout the genome and are usually associated with the promoters of genes. Methylation of the CpG island occurs on cytosines. Unmethylated promoters are generally found in active genes, whereas inactive genes usually have a methylated promoter region. This mechanism is involved in normal human development, X-chromosome inactivation and imprinting.⁷⁷ With respect to imprinting of the PWS region on chromosome 15, the maternal chromosome is normally methylated and thus inactive, whereas the paternal chromosome is unmethylated and active. The inheritance of one methylated, inactive PWS locus and one unmethylated, active PWS locus is crucial for normal human development.

Southern Blot Analysis

Initially, genetic testing for PWS involved chromosome analysis and DNA analysis of several restriction fragment length polymorphisms (RFLPs) and microsatellites to identify deletions and UPD. This was time consuming and not useful for some families due to a lack of informative polymorphic markers. Furthermore, high-resolution karyotypes were unreliable for the detection of a PWS deletion.⁶⁶

The ﬁ rst clinically available DNA test for Prader-Willi syndrome involved Southern blot analysis, a method still used in many molecular diagnostic laboratories. Southern blot analysis involves several steps, the ﬁ rst of which is digesting DNA with restriction enzymes. The restriction fragments are then separated on the basis of size by agarose gel electrophoresis, with the smaller fragments migrating through the gel faster than larger ones. The DNA is then denatured to produce single stranded fragments that are transferred to a nitrocellulose or nylon membrane (Southern blot) on which they become immobilized.

The single stranded DNA on the Southern blot is then hybridized to a single stranded DNA probe (usually several hundred base pairs to several kilobases in length) that has been radioactively labeled. The single stranded DNA probe binds to its complementary single stranded

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Table 4.1. Comparison of Methods Used for the Molecular Genetic Diagnosis of Prader-Willi Syndrome Method Advantages Disadvantages Southern blot >99% detection rate* Requires 5 mg of DNA Simple to perform Limited by enzyme restriction sites analyzed Reliable, extensively used Does not determine etiology of PWS* Radioactivity required 2–3 week turnaround time False positives due to incomplete restriction enzyme digestion mPCR Nonisotopic Bisulfi te treatment of DNA is time consuming Avoids pitfall of restriction site polymorphisms Need to adjust relative primer concentration to 3–5 day turnaround time achieve amplicons of similar intensity ~200 ng of DNA required Fluorescence melting Nonisotopic Bisulfi te treatment of DNA required curve analysis 2–3 day turnaround time Expensive equipment No transfer of PCR products or electrophoresis involved Appropriate primer design is necessary ~200 ng of DNA required BRA£100 ng of DNAMore labor intensive than mPCR No concerns regarding preferential amplifi cation of PCR Risk of false negative results in PWS due to products incomplete digestion Methylation- Rapid turnaround time Limited by enzyme restriction sites analyzed sensitive restriction Nonisotopic Risk of false negative results in PWS due to digest followed by PCR No bisulfi te DNA treatment required incomplete digestion 500 ng DNA required Labor intensive (2 digests and 3 multiplex PCRs per sample) SNRPN expression Nonisotopic Not diagnostic for AS analysis (RT-PCR) 1–2 day turnaround time RNA is more labile than DNA Should detect 100% of PWS, regardless of etiology (compared Special laboratory handling required for RNA with a >99% detection rate for the above methods) * None of the methods included in this table will determine the PWS etiology, and all will detect >99% of subjects with PWS. Note: Fluorescence in situ hybridization (FISH) is not included as a molecular genetics technique.

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DNA sequence on the nylon membrane. Excess probe is then washed off of the blot, and the membrane is exposed to X-ray ﬁ lm, a process called autoradiography. The ﬁ lm is then developed, with the autoradiogram revealing the location on the original agarose gel where the probe was bound, or hybridized, corresponding to the allele sizes of the restriction fragments.

Southern blot analysis for the diagnosis of PWS requires the use of two restriction enzymes, one that will cleave DNA regardless of the methylation status of the DNA (maternal and paternal chromosome 15), and another enzyme that will only cleave its recognition sequence if the DNA is unmethylated (paternal chromosome 15). Many different probes have been used for the diagnosis of PWS including the zinc ﬁ nger gene, ZNF127 (D15S9), PW71B (D15S63), and the gene for the small nuclear ribonucleoprotein N (SNRPN) exon 1 (summarized by Buchholz et al.⁵). Southern blots hybridized with the ZNF127 probe are difﬁ cult to interpret and thus are not used in most clinical laboratories.

PW71B and SNRPN have been used extensively in clinical laboratories because they give a clear difference between the maternal and paternal alleles.

Methylation analysis by Southern blotting using either the PW71B or SNRPN probes will detect over 99% of subjects with PWS.⁴ The SNRPN and PW71B probes have been validated as diagnostic tests for PWS.⁴² Few subjects have been reported in the literature with typical PWS features and normal methylation studies. One example involved an individual with a classic PWS phenotype, normal SNRPN and PW71B methylation studies, and normal UPD studies. Chromosome Figure 4.1. Laboratory testing for diagnosis and characterization of Prader- Willi syndrome.

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analysis detected a de novo balanced translocation involving 15q11–q13 disrupting the SNRPN locus, which was conﬁ rmed by SNRPN expres- sion studies.⁶⁷

Disadvantages of Southern blot analysis include: a large amount of high quality, high molecular weight DNA must be used (usually ~5 mg);

and information is only provided about those methylated regions within the PWS sequence recognized by methylation sensitive restriction enzymes. In addition, radioactivity is usually involved, although nonradioactive Southern blotting using chemiluminescent detection has been described.⁶⁹ Furthermore, because the methylation test does not distinguish among deletions, uniparental disomy, and IC defects, reﬂ ex testing is required to deﬁ ne the etiology of the PWS. The PW71B and SNRPN probes are reliable as the primary approach to PWS molecular diagnosis. However, PWS patients have been reported with dis- cordant results with the two probes.^5,6 Thus, for PWS patients with a classic phenotype who have a negative methylation result using one probe, studies using either the opposite probe or the same probe with different restriction enzymes should be performed.

PW71B

PW71, located at D15S63, is an imprinted locus of unknown function that resides within the PWS/AS critical region. The probe, PW71B, used for methylation studies²⁴ is a 365bp HaeIII fragment cloned into the SmaI site of the plasmic vector, pUC19. The probe is excised by digestion with EcoRI and HaeIII and is available through the American Type Culture Collection (ATCC/NIH Repository #99412/99413).

PW71B includes the original PW71 probe²⁶ and gives better hybridization signals than the original probe.²⁴ At least one HpaII and one CfoI restriction enzyme recognition sequence are methylated on the maternal, but not paternal, chromosome 15. Combinations of restriction enzymes that can be used with PW71B include BglII/CfoI, HindIII/

CfoI, and HindIII/HpaII. The maternal and paternal fragment sizes vary based on the combination of restriction endonucleases used (Figure 4.2).

The PW71B methylation test accurately detects most cases of PWS²⁹; however, false positive and false negative results can occur as well as technical diffi culties in the laboratory.^6,8,48,72 Technical diffi culties reported with PW71B are due to the probe being so small. This can be resolved by using a high probe count, low stringency washes, and dextran sulfate, which increases the hybridization signal by preventing nonspecifi c binding of the probe to the membrane.^24,25 False positives may occur due to incomplete digestion with the methylation sensitive restriction enzyme.²⁵ To control for the completeness of enzyme digestion, the laboratory should run a normal sample in addition to samples positive for AS and PWS on all Southern blots.

Unusual results have been reported due to a benign polymorphism of North-African origin in the restriction enzyme recognition sequence for BglII.²³ False positive results reported by Buiting et al.⁶ were due to a 28 kb deletion spanning the D15S63 locus with a frequency of 1 in 75

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among the Ashkenazi Jewish population.⁶⁴ This deletion is a benign variant with no effect on imprinting in the PWS region; it arose due to a recombination event between repetitive Alu sequences. False negative results for PW71B and SNRPN have been reported due to mosaicism for a 15q11–q13 deletion, detected by FISH studies but masked in methylation studies by a normal cell population.⁴⁸

SNRPN

Exons 1–3 within the SNRPN upstream reading frame encode a protein product of unknown function termed “SNURF” (SNRPN upstream reading frame). Exons 4–10 encode SNRPN (SmN), which functions in mRNA splicing. The SNRPN gene is expressed only from the paternal allele³²; there is extensive methylation of the maternal SNRPN CpG island, which represses transcription of SNRPN from this allele. This differential methylation is the basis of the SNRPN molecular diagnostic test for PWS.^33,68 The CpG island of the SNRPN gene contains several NotI sites that are methylated on the maternal chromosome. The probe used for analysis is a 0.9 kb NotI fragment cut from a 4.2 kb XbaI fragment containing SNRPN CpG island and is available from the American Type Culture Collection (ATCC Repository #95678, #95679).

Following digestion of genomic DNA with NotI and XbaI, Southern blot analysis of a normal maternal chromosome results in a 4.2 kb band, Figure 4.2 A. Restriction map and RFLP sizes surrounding PW71B probe23,24,25,29

(*MT-sensitive site, methylated site will not cut with enzyme). B. Autoradiogram of Southern blot using the probe PW71B with HpaII and HindIII digested DNA. The patient analyzed is negative for Prader-Willi syndrome (PWS); both DNA bands are present. AS = Angelman syndrome.

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consistent with the methylation of the NotI sites (which will not cut with the enzyme). A normal paternal chromosome results in a 0.9 kb band, because the unmethylated NotI sites are cut by the enzyme (Figure 4.3).

For SNRPN Southern blot analysis, blood should be collected in either EDTA or ACD tubes. DNA extracted from blood collected in sodium heparin tubes may not be completely digested with NotI, resulting in a weak paternal band and a possible false positive result for PWS.⁴⁰ In addition to a weak paternal band, additional bands larger than the expected 4.2 kb band are also seen in DNA extracted from sodium heparinized blood samples. Studies to determine the completeness of the digest can be performed if the laboratory suspects incomplete NotI digestion.^25,40

Polymerase Chain Reaction (PCR)

Several methods involving PCR analysis for the molecular diagnosis of PWS have been developed, including methylation-specifi c PCR, fl uorescence melting curve analysis, bisulfi te restriction analysis, and reverse transcription PCR. These techniques have several advantages

Figure 4.3 A. XbaI/NotI restriction map and RFLP sizes surrounding SNRPN probe⁶⁸ (*MT-sensitive site, methylated site will not cut with enzyme). B. Autoradiogram of Southern blot using the probe SNRPN with NotI and XbaI digested DNA. The patient analyzed is negative for Prader-Willi syndrome (PWS); both the 4.2 kb and the 0.9 kb DNA bands are present. AS = Angelman syndrome.

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over Southern blot analysis for the diagnosis of PWS (Table 4.1), including a rapid turnaround time and smaller amount of DNA template required.³⁹ As with Southern blot analysis, each method will detect virtually all cases of PWS.

Methylation-Speciﬁ c PCR (mPCR)

In 1997, Zeschnigk and co-workers demonstrated that >95% of the CpG dinucleotides around SNRPN exon 1 are methylated on the maternal chromosome and not methylated on the paternal chromosome. The template for methylation-PCR (mPCR) is bisulfi te treated DNA. Bisul- fi te converts unmethylated cytosines to uracil.³⁵ PCR primers specifi c to the modifi ed and unmodifi ed DNA sequence were designed to distinguish between the maternal and paternal alleles.39,41,78,79

Following an overnight incubation of DNA treatment with bisulﬁ te, multiplex PCR using maternal- and paternal-speciﬁ c primers analyzed by gel electrophoresis produces two different amplicons, one corresponding to the maternal allele and the other corresponding to the paternal allele. The amplicon sizes vary depending on the primers used. Using the primers described by Kubota et al.,⁴¹ normal individuals show a paternal product of 100 bp and a maternal product of 174 bp, whereas PWS patients show only the maternal 174 bp product and AS patients show only the 100 bp paternal product (Figure 4.4). Untreated

Figure 4.4. Methylation-speciﬁ c PCR (mPCR) analysis of the SNRPN promoter using method described by Kubota et al.⁴¹ Lane 1 (far left): size standard (marker V, Boehringer Mannheim). Lane 2: no DNA or negative control. Lanes 3–4: patients analyzed for Prader-Willi syndrome (PWS); both are normal with the maternal (174 bp) and paternal (100 bp) bands or amplicons present. Lanes 5–6: normal controls. Lane 7: PWS positive control. Lane 8: AS positive control.

Lane 9 (far right): no DNA or negative control.

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DNA does not result in a PCR product since the primers are not complementary to the unmodiﬁ ed DNA sequence.⁴¹ Denaturing high per- formance liquid chromatography (DHPLC) and primer extension ion pair reverse phase HPLC are alternative methods to gel electrophoresis for the analysis of mPCR products.^2,49

PCR using bisulfi te treated DNA produces accurate results in PWS patients, in complete agreement with Southern blot analysis.^11,54,79 This method requires only a small amount of DNA template, which can be extracted from whole blood, lymphoblasts, dried blood cards, cultured amniocytes, cultured chorionic villi, heparinized blood, and archived fi xed-cell suspensions remaining from FISH studies.^41,75 This method analyzes the methylation status of the SNRPN gene along many CpG dinucleotides, avoiding the potential pitfall of restriction site polymorphisms²³ and incomplete digests. One drawback of mPCR is that the smaller, paternal allele is preferentially amplifi ed in this assay, which may result in a false negative result for PWS or a false positive result for AS. To obtain amplicons of similar intensity, the relative primer concentrations need to be adjusted with three- to fourfold less paternal primers than maternal primers.^41,79 Skewed signal intensity may also refl ect mosaicism for UPD, which can be confi rmed by microsatellite analysis of individual cells.³⁶

Fluorescence Melting Curve Analysis

Bisulfi te treated genomic DNA amplifi ed with primers specifi c for SNRPN can also be analyzed by melting curve analysis.⁷⁷ This method is based on the difference in melting temperatures between the maternal and paternal PCR amplicons. Double-stranded DNA denatures at a temperature dependent on its sequence, with G-C base pairs melting at a higher temperature than A-T base pairs. Using this technology, amplifi cation is performed in a thermal cycler connected to a fl uorom- eter or LightCycler, eliminating the need for gel electrophoresis. This method has all of the benefi ts of mPCR with an even shorter turnaround time. However, careful primer design is essential to a successful assay, and this method utilizes expensive equipment that may not be available in all clinical molecular laboratories.

PCR Using DNA Digested with Methylation-Sensitive Restriction Enzymes

A variant of mPCR involves the digestion of genomic DNA with methylation-sensitive restriction enzymes followed by PCR amplifi cation of the SNRPN promoter.²¹ Two separate digests are performed: one with NotI, which will only digest unmethylated DNA; and another with McrBC, which will only digest methylated DNA. Three separate multiplex PCRs are then performed using uncut DNA, NotI treated DNA, and McrBC treated DNA as templates. Each PCR contains two sets of primers, one specifi c for SNRPN and another control primer set specifi c for a nonimprinted gene on another chromosome. SNRPN sequences are not amplifi ed from McrBC treated DNA from PWS patients or from NotI treated DNA from AS patients. Though reported to be 100% sensitive and specifi c for PWS and AS caused by deletions and UPD, this assay will only detect CpG methylation that occurs in methylation-

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sensitive restriction sites and is prone to false negative results second- ary to incomplete NotI digestion. This method is nonisotopic, requires a minimal amount of DNA, and does not require bisulﬁ te treatment of the DNA.

Bisulﬁ te Restriction Analysis (BRA)

Bisulfi te restriction analysis (BRA) was developed as an alternative to mPCR.^73,74 This method involves two rounds of methylation- independent nested PCR amplifi cation of bisulfi te treated genomic DNA, followed by CfoI digestion. Bisulfi te treatment destroys the CfoI recognition sequence on the paternal allele, so that the paternal amplicon will remain undigested whereas the maternal allele will be cleaved. There is the risk of a false negative result in a PWS patient due to incomplete digestion, and BRA is more labor intensive than mPCR, requiring the bisulfi te treatment of DNA, two separate PCRs, a restriction enzyme digest, and gel electrophoresis. However, BRA eliminates the concerns of preferential amplifi cation of the paternal allele in mPCR and can also be used on fi xed-cell suspensions remaining from cytogenetic studies.⁷⁴

Reverse Transcription PCR (RT-PCR)

Although Southern blot, mPCR and its variants will detect >99% of PWS cases, a few cases have been reported in the literature with a classic PWS phenotype, normal methylation studies, and balanced 15q11q13 translocations.22,43,63,67 In these cases, disruption of the SNRPN locus by the translocation was revealed by abnormal SNRPN expres- sion. Expression studies of the SNRPN gene are used as another means for the clinical molecular diagnosis of PWS.³² The SNRPN expression test is rapid and reliable in the molecular diagnosis of PWS.^18,76 This assay involves the reverse transcription of RNA extracted from blood collected in either sodium EDTA or sodium heparin tubes. Multiplex- PCR is performed on the cDNA product with two sets of primers, one speciﬁ c for the SNRPN mRNA and one speciﬁ c for a constitutively expressed X-linked gene (WASP). The products are analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide.

Absence of the SNRPN product in the presence of the control amplicon indicates lack of SNRPN expression, hence a diagnosis of PWS.

Similar to mPCR, RT-PCR is nonisotopic, requires only a small amount of blood, and has a short turnaround time (1–2 days). RT-PCR is predicted to detect all cases of PWS, regardless of etiology, but is not informative in diagnosing AS. This method has been shown to correctly identify cases of PWS due to deletion, UPD, imprinting defects, and translocations (including those that are not detectable by standard methylation analysis), although relatively few cases of PWS due to IC microdeletions and translocations have been studied. RT-PCR involves the isolation and handling of RNA, which requires dedicated “RNAse free” laboratory areas. Another drawback of working with RNA is that it degrades more rapidly than DNA. False positive or negative results have not been reported using RT-PCR; however, theoretically a false negative result could occur in a patient with normal levels of nonfunc- tional SNRPN protein.¹⁸

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Uniparental Disomy

Once the diagnosis of PWS has been established using SNRPN methylation or expression analysis, additional studies are needed to determine the molecular mechanism of PWS, provide accurate genetic counseling, and establish recurrence risks for other family members (Figure 4.1). Over 70% of subjects with PWS have deletions; therefore, FISH studies should be the initial refl ex test performed. If FISH studies are negative (normal) then the possibility of UPD, accounting for about 20% to 25% of PWS subjects, should be considered.⁴⁴ Uniparental disomy studies involve the amplifi cation of multiple microsatellites within the PWS critical region as well as microsatellites outside of the critical region (to distinguish between an IC deletion and UPD). DNA from the proband and both parents is required for UPD analysis. If only one maternal allele is present within the critical region as well as proximal and distal loci, isodisomy is implied. If both maternal alleles are present, heterodisomy is implied. Maternal inheritance within the critical region, with maternal and paternal alleles present in the outside regions, will occur if a deletion is present. A number of microsatellites within and adjacent to the critical region can be used for this analysis.4,7,15,19,28,30,47,55 Information regarding many of the microsatellites is available on the Genome Database Web site (www.gdb.org²⁷), including primer sequences and references. When possible, microsatellites can be amplifi ed by multiplex PCR to save time and reagents. The PCR products can be radioactively labeled and analyzed by polyacrylamide gel electrophoresis. Alternatively, microsatellites can be amplifi ed by multiplex PCR using fl uorescence labeled primers and analyzed on an automated DNA sequencer.⁴⁶

Imprinting Defects

PWS patients with positive methylation DNA studies, a normal karyo- type and FISH studies, and biparental inheritance of chromosome 15 are assumed to have an imprinting defect. This occurs in less than 5%

of PWS cases. Approximately 15% of the imprinting defects are due to microdeletions within the IC, the majority of which are inherited, with a 50% recurrence risk.^8,9,10,57 An abnormal methylation pattern in the unaffected father of a PWS proband is indicative of a familial IC deletion. Methylation studies of a carrier father will reveal an AS methylation pattern. A normal methylation pattern in the father of a PWS proband is suggestive of a nondeletion case, which accounts for the other 85% of PWS imprinting defects.^8,9

Nondeletion imprinting defects occur during spermatogenesis when the imprint on the father’s maternally inherited chromosome 15 is not erased.^7,9 Although the proband inherits one chromosome 15 from each parent, the paternally inherited chromosome carries the grandmaternal imprint, thus the proband has two maternally imprinted chromosomes.⁵⁰ The mechanism for this type of defect is not known. No IC point mutations have been identiﬁ ed in patients with a nondeletion imprinting defect.^7,9,57

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High-Resolution FISH Probes

Tharapel et al.⁷⁰ reported their use of primed in situ labeling (PRINS) to identify deletions of SNRPN, GABRB3, and DGCR2/TUPLE1. This method has potential use to identify small deletions within the PWS imprinting center.

High-resolution FISH probes to test for constitutional and acquired chromosome imbalances have been developed.^38,62 The probes can identify small deletions involving only the imprinting center, including those that have been reported in multiplex PWS families. Their PWS- SRO probe performed as expected in typical PWS deletion cases and in a case of familial PWS in which a deletion was conﬁ ned to the PWS imprinting center.³⁸

Prenatal Diagnosis

Prenatal diagnosis of PWS and genetic counseling should be offered when the father is a microdeletion carrier, as the recurrence risk for PWS in this situation is 50%. A carrier female is not at risk to have children with PWS; however, she is at risk to have affected grandchil- dren through her carrier sons. Prenatal testing is also indicated for PWS due to a paternal translocation, as there is an increased recurrence risk.

For PWS due to large deletions, nondeletion IC defects, and UPD, the recurrence risk is small (<1%); however, prenatal testing may be offered to reassure the parents.¹

PWS prenatal testing should be considered in women undergoing routine prenatal testing for fetal chromosome abnormalities by CVS or amniocentesis when any of the following are detected: 15q deletions, mosaic trisomy 15, familial or de novo translocations or marker chromosomes involving chromosome 15 (including isochromosome 15).^1,44,60,65 All of these situations are associated with an increased risk of 15q deletions or UPD.

Microsatellite analysis, which requires fetal as well as parental DNA, can be used to test for UPD or small deletions. Microsatellite analysis may also be used for preimplantation genetic diagnosis to detect PWS IC deletions. Methylation analysis can be performed on fetal tissue to detect >99% of PWS cases. PW71B is known to be hypomethylated in amniocytes and chorionic villi, which could result in a false positive diagnosis of AS. ZNF127 and PW71B are not diagnostic for prenatal specimens.24,31,42,65,72 SNRPN analysis by mPCR or Southern blot analysis is the method of choice for prenatal PWS diagnosis.^4,42

Selecting a Reference Laboratory

The American College of Medical Genetics (ACMG) and American Society of Human Genetics (ASHG) have recommended that PWS/AS genetic testing include either: (1) Southern hybridization with SNRPN or PW71B probes followed by FISH, UPD, or imprinting studies in positive cases, or (2) FISH and methylation analysis performed simultane-

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ously, followed by UPD studies if needed.¹ In either scenario, chromosome analysis is recommended to identify a translocation or other abnormality. It is recommended that the methods used take into account a number of factors, including the availability of testing, known test results for a given patient, and physician experience. When the like- lihood that a patient will test positive for PWS is less than 50%, it is less expensive to perform DNA methylation studies, with FISH and UPD studies done only if PWS or AS is diagnosed.⁵³ However, whenever an expedient diagnosis is crucial—for instance, in a hypotonic newborn—

simultaneous FISH and DNA testing should be considered.

We surveyed U.S. clinical molecular genetics laboratories listed on the GeneTests web site (www.geneclinics.org²⁰) and found that about half perform PWS testing by PCR methods and half utilize Southern blot analysis, with SNRPN being the most common probe used (K.G.

Monaghan, unpublished data). About one third of the labs offer microsatellite analysis for UPD studies, and two thirds offer FISH for PWS. Approximately half of the labs offer prenatal diagnosis for PWS. This is consistent with a recent American College of Medical Genetics/College of American Pathologists (ACMG/CAP) molecular gen etics survey (MGL-A, 2003) in which 46% of labs reported using Southern blot analysis, 46% used PCR, 4% performed SNRPN expression studies by RT-PCR, and a minority used a combination of methods including Southern blot and PCR or Southern blot and SNRPN expression studies.

Laboratories in the U.S. can be inspected and accredited by the CAP laboratory accreditation program and Clinical Laboratory Improve- ment Act (CLIA). In addition, profi ciency testing is available for many genetic conditions, including cytogenetics, FISH, and PWS/AS DNA testing. Of the labs participating in a recent MGL-CAP survey, 93% (43/46) correctly genotyped the PWS/AS specimens included in the profi ciency test. In the 2002 CY-C CAP survey, all participating laboratories correctly scored the PWS unknown case. Although participation in inspections and profi ciency testing do not exclude the possibility of technical or clerical errors, participation in these voluntary inspections and lab surveys indicates the laboratory’s commitment to quality.

Other considerations when selecting a reference laboratory include the cost and reporting time. In addition to the many technical advantages of mPCR (Table 4.1), our survey found that PCR offers a short turnaround time (3–5 days) and a lower price (~US$250–$400) compared with Southern blot analysis (2–3 weeks; ~US$300–$500) and FISH (3–5 days; ~US$300–$500).

Finally, when a laboratory report is inconsistent with the clinical presentation of a patient, it is important to keep in mind the pitfalls of genetic testing. An abnormal result can be masked by polymorphisms within restriction enzyme recognition sequences (refer to section on Southern blotting), mosaicism for 15q11–q13 deletions,⁴⁸ or mosaicism for UPD.³⁶ Incomplete restriction enzyme digests can result in a false positive for PWS, while degraded DNA can result in a false positive for AS when analyzed by Southern blotting. A normal polymorphism within a primer sequence can interfere with PCR ampliﬁ cation. Sample

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mix-ups can occur in the clinic or in the laboratory. Testing for the wrong disease can result in a noninformative negative result, in which case the differential diagnosis should be considered. Mistaken pater- nity will yield uninformative results for microsatellite analysis. Con- tamination of samples is especially problematic for PCR-based assays and contamination of fetal specimens with maternal DNA can result in inaccurate prenatal results. Laboratories should perform additional studies to exclude maternal cell contamination of any prenatal specimen. If there is any doubt in the patient’s result it is reasonable to repeat testing using an alternative methodology or to request a second specimen.

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