A deletion in the paternal 15q11.2-q13 chromosomal region is the most common cause of PWS; however, it has become clear that Type 1 and 2 deletions are not the only type of deletions that occur in individuals with PWS. Among our cohort of 88 deletion subjects with PWS, we found 36.4% with Type 1 deletion, 55.7% with Type 2 deletion and 8% with a unique or an atypical deletion. The higher number of Type 2 vs
Type 1 is similar to the findings of other studies.15, 16, 17
The prevalence of unique or atypical deletions was higher than what we expected and has not previously been reported for PWS, but was comparable to the prevalence rate (9.1%) found in AS.7
The 15q11.2-q13 region is highly vulnerable to structural rearrangements, such as deletions, duplications, supernumerary marker chromosomes, and translocations due to presence of LCRs in the region.24, 25
In our subjects with unique or atypical deletion sizes, two subjects (cases 6 and 7) had both proximal and distal BPs involving LCRs (ie, BP2 and BP5). Another three of our subjects (cases 1, 2 and 5) had a proximal BP in an LCR (ie, either BP1 or BP2), but the distal BP was unique and located proximal to BP3 in an interval between ATP10A
. Cases 3 and 4 had an unbalanced translocation with neither BP involving an LCR in the 15q11.2-q13.3 region.
In this study, we also examined genotype–phenotype relationships in our seven subjects with unique or atypical deletion sizes. Three of our subjects (cases 1, 3 and 4) with an intact OCA2
gene had normal skin pigmentation comparable to other members of their family. This is not surprising given the role of OCA2
in skin pigmentation.26
Case 2 came to our attention only after his brain was donated to our brain bank, so we cannot comment on his skin pigmentation. The clinic notes we obtained have no mention of his pigmentation. Case 5 had a deletion within intron 18 of OCA2
and was hypopigmented. Case 6 and 7 had large deletions that included OCA2
, but hypopigmentation was not noted. In our clinics, we have found that hypopigmentation in PWS deletion cases is much harder to discern in the more deeply pigmented ethnic and racial groups. Case 6 is Hispanic and case 7 is Black, which probably explains why hypopigmentation was not appreciated in these two subjects.
One subject had a much smaller deletion (2.46
Mb) than a typical deletion (Case 1). His deletion spared the GABAA
receptor subunit gene clusters and OCA2
. Interestingly, he had an above average birth weight, macrocephaly, large hands/feet and tall stature for PWS (before he started growth hormone), lower-than-average pain tolerance, and somewhat delayed onset of the voracious appetite and food-stealing behavior. These atypical features contributed to the delay in his PWS diagnosis. However, the moderate skin picking behavior in this subject was rather puzzling. Given the postulated role of GABA in the mediation and perception of pain,27
we would have anticipated that sparing the GABAA
receptor subunit gene clusters would have lessened this behavior. In addition, he manifested the common behavioral characteristics of individuals with PWS including temper tantrums, easily angered and rigidness. Furthermore, his cognitive IQ was 63, well within the typical range for PWS.28
Unfortunately, we do not have detailed behavioral information on case 2 whose deletion also spared the GABAA
receptor subunit gene clusters. Therefore, based only on this one subject, it would appear that sparing the GABAA
receptor subunit gene clusters does not alter the typical PWS neurobehavior.
Interestingly our two subjects (cases 6 and 7) with large deletions spanning >9
Mb between BP2 and BP5 had certain similarities and differences in their phenotypic presentation. In both the CHRNA7
gene was deleted, but case 7 had an additional 250
kb deletion telomeric to CHRNA7
involving the DKFZp434L187
encodes the alpha-7 subunit of the neuronal nicotinic acetylcholine receptor.29
Recently several groups have implicated CHRNA7
as a candidate gene for the 15q13.3 microdeletion syndrome whose clinical manifestations include facial and digital dysmorphology, expressive language deficit, and various neuropsychiatric disorders, such as schizophrenia, autism, epilepsy and mental retardation.30, 31, 32, 33
The finding that cases 6 and 7 had large deletions encompassing the 15q13.3 region prompted us to request an EEG be done on both of them. Similar to those with the 15q13.3 microdeletion, case 6 had both clinical and EEG-documented seizure activities. Surprisingly, her parents, teachers and health care providers were unaware of her absence seizures until her abnormal seizure related behavior (eg, looking away, lip smacking, etc.) correlated with the EEG wave changes. Case 7 also had an abnormal EEG; however no clinical seizure activity has been noted in this subject. As he is still young, it would be reasonable to assume that he is at increased risk for seizure activities. In addition, both case 6 and 7 lacked the typical facial features of PWS, but did share some facial features in common with the 15q13.3 microdeletion subjects ().
Figure 2 The two PWS subjects with large atypical deletions and lacking the typical PWS facial gestalt. Case 6 at 9 months (a), years (b) and 7 years (c, d). At 9 months (a) she appears well nourished with a weight for length at the 75th percentile. Features (more ...)
Although sharing some similarities, case 6 and 7 did have drastic differences in their clinical manifestations. Case 6 had a much milder postnatal course, less severe developmental delay, macrocephaly, and higher intellectual and academic functioning. At 7.2 years she had a cognitive IQ score of 89 and an achievement score of 109 both of which are significantly higher than typically found in PWS.28
This is surprising as her deletion encompasses both the PWS region as well as the 15q13.3 microdeletion region. In contrast, case 7 had a much more severe failure-to-thrive phase requiring a feeding tube for 2.5 years, microcephaly, markedly delayed gross and fine motor skills as well as speech development. At 4.9 years he was still not walking independently. Case 7's clinical findings are more in line with what we would have expected from deleting two microdeletion syndrome regions. At this point it is not clear why there is such a dramatic difference between cases 6 and 7 who share a similar-sized deletion of ~9
Mb. There are several possibilities for the differences. First, the genetic variation in the 15q13.3 region on the maternal chromosome may become ‘unmasked' by the paternally derived deletion, which may have contributed to the more severe clinical phenotype in case 7. Second, genetic variation elsewhere in the genome may have contributed to the composite clinical features in case 7. A great deal of variability in clinical features has been described for individuals with the 15q13.3 microdeletion including some parents with the deletion who lack the clinical features found in their offspring.33
Furthermore, the 15q13.3 region, like the 22q11.2 and 16p12.1 regions, may be another example of a ‘susceptibility region' that requires a ‘second hit' elsewhere in the genome to become fully penetrant as a neurodevelopmental phenotype.34, 35
Third, the DKFZp434L187
transcript (deleted in case 7) downstream to CHRNA7
may require further evaluation for its potential role in the more severe developmental delay and failure-to-thrive, although this is very unlikely given that this transcript has not been shown to code for any important function to our knowledge.
We identified several strengths and weaknesses of the MS-MLPA technique. MS-MLPA reliably detected the deletion as evidenced by 100% agreement with our laboratory data and aCGH. It also provided more detailed information regarding the size of the deletion than standard FISH due to many (25–32) probes across the 15q11.2-q13 region. Thus, the MS-MLPA technique allows for a reasonable approximation of the BPs and deletion size. Compared with aCGH, the MS-MLPA technique was much more labor/cost-effective, although aCGH provides more precise information regarding the extent of the deletion. In addition, the DNA methylation component of MS-MLPA allows differentiation between PWS and AS deletions, and maternal and paternal 15q11.2 duplications, as well as between uniparental and biparental disomy. Therefore, a compelling argument could be made that MS-MLPA should be the first test used when contemplating AS or PWS as a possible diagnosis, especially since important genotype–phenotype correlations will likely be forthcoming.
In this study, we had an opportunity to evaluate both the A1 and B1 versions of the commercial MS-MLPA kit. The A1 version has two probes for OCA2, which are useful to identify unique deletions with distal BPs between GABRB3 and BP3. If we had not used the A1 version, we would have missed the unique deletion sizes in cases 3, 4 and 5. The B1 version, on the other hand, is designed to provide additional coverage at the bipartite AS and PWS imprinting center and SNORD116 regions. Taken together, the A1 version appears superior in detecting deletion sizes of AS and PWS, whereas the B1 version is superior in identifying small deletions in the imprinting center and/or the SNORD116 region.
In conclusion, this study makes several important points. First, deletions should be characterized by accurately determining both their proximal and distal BPs, rather than just their proximal BP. The designation of Type 1 deletion should be strictly reserved for those deletions between BP1 and BP3, and Type 2 deletion for those deletions between BP2 and BP3. This is important for genotype–phenotype counseling in the future, as unique or atypical (ie, not type 1 or 2) deletions due to a novel distal and/or proximal BP could result in a milder or more severe phenotype than either Type 1 or Type 2 deletions. This may also contribute to the lack of current consensus in genotype–phenotype comparisons of Type 1 vs Type 2 deletions, as the distal BPs were not always well delineated in many of these studies. Second, the frequency of deletions that are neither Type 1 nor 2 (8% in our study) is higher than previously recognized in PWS. Third, genotype–phenotype studies on individuals with unique or atypical deletions have the potential to elucidate the role of the various genes in the 15q11.2 region. Fourth, the mechanisms for producing unique deletions need further clarification as at least five of the subjects in this study had at least one BP that did not occur at a 15q11.2-q13 LCR region. Fifth, the differences in clinical manifestations of our two subjects with a deletion extending into the 15q13.3 region further underscore the clinical variability of the 15q13.3 microdeletion syndrome. In our future studies, we plan to obtain more detailed phenotypic information using specific neurobehavioral measures, such as cognitive and behavioral profiles, comorbid psychiatric illnesses, and response to certain medication, to more fully examine genotype–phenotype relationships in these subjects.