Prader–Willi syndrome (PWS, OMIM 176270) and Angelman syndrome (AS, OMIM 105830) are caused by the loss of expression of imprinted genes at 15q11-q13 [1]. PWS results from the absence of the paternal allele of 15q11-q13, whereas AS results from the absence of the maternal allele in the same region [2]. This phenomenon is called genomic imprinting. PWS and AS occur in one in 10,000–30,000 live births [3].

Both syndromes are neurodevelopmental disorders; however, their clinical phenotypes differ [4]. PWS is characterized by neonatal hypotonia, feeding problems, failure to thrive, hypogonadism, and childhood-onset obesity [5]. AS patients present with seizures, microcephaly, and severe developmental delay [6]. When a neonate shows neonatal hypotonia or developmental delay, PWS or AS should be considered as a part of the differential diagnosis.

Several molecular mechanisms lead to PWS and AS: deletion, uniparental disomy (UPD), imprinting defect (ID), and balanced translocation [7]. Deletion of 15q11-q13 accounts for approximately 70% of cases and is the leading cause of both syndromes. Deletions are subdivided into typical type I or II deletion, which respectively range from breakpoint (BP)1 to BP3 or from BP2 to BP3, and atypical deletion [8]. UPD is mostly due to maternal meiotic non-disjunction and accounts for 3%–30% of cases, whereas ID causes 1%–5% [9]. Loss of UBE3A function causes AS in 10%–20% of patients [10].

Because the molecular mechanisms of PWS and AS determine the recurrence risk, prognosis, and clinical phenotypes, understanding the genetic profiles of these diseases can help clinicians make an accurate diagnosis and counsel patients and their families appropriately [11]. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a diagnostic method for the simultaneous detection of copy number abnormalities and methylation status [12]. It can distinguish deletional types from non-deletional types of PWS and AS. We evaluated the clinical utility of MS-MLPA in comparison with that of methylation-specific (MS)-PCR in Korean PWS and AS patients. In addition, we investigated the relationship between clinical phenotypes and molecular mechanisms determined by MS-MLPA.

Chromosomal microarray (CMA) is currently considered the first-tier diagnostic genetic test for neurodevelopmental disorders [14, 15]. However, there remains a necessity for methylation analysis, especially for imprinting disorders, because CMA is not sufficient for diagnosis of these disorders [16]. We showed that MS-MLPA can not only diagnose PWS and AS, but also reveal the underlying molecular mechanisms. We also demonstrated the relationship between molecular mechanisms and clinical characteristics of Korean PWS and AS patients. Our findings support that MS-MLPA is a useful diagnostic test for PWS and AS.

We detected the deletion type in 57.8% of PWS cases, which is in contrast to results in a previous Korean study in 2004, in which deletion accounted for 80% of PWS cases [17]. In line with our results, recent studies have demonstrated that the deletion type in PWS constitutes approximately 60% of total cases [18, 19]. Butler, et al. [18] reported that in PWS, the maternal age was higher in UPD cases than in deletion cases. Although we were not able to assess maternal age for all patients, the average maternal age in Korea is increasing yearly, from 30.0 years in 2004 to 32.4 years in 2016, according to the Korea Statistics [20]. Thus, the increase in the non-deletion type may be because UPD is more prone to occur as maternal age increases.

PWS patients with type I deletions show a more severe phenotype than those with type II deletions [21]; however, there were no significant differences in the diagnostic scores of our patients according to the deletion range. The mean Holm score of total PWS was even lower than the diagnostic score. This may be explained by poor clinical evaluation or diagnosis before clinical symptoms present due to advanced molecular diagnosis.

An atypical deletion, ranging from SNRPN to GABRB3, was detected in one PWS patient (case 39). Additional CMA results confirmed that the patient had a 2.8 Mb deletion from PWRN2 to GABRB3. He visited our hospital for lymph node enlargement at 12 years of age. At that time, he had mild learning disabilities and obesity [22, 23]. His Holm score was 5, which is higher than the mean score of PWS patients. This deletion type showed that MKRN3, MAGEL2, NDN, and C15orf2 are not indispensable for PWS symptoms [24].

One AS patient with an IC deletion (case 66) visited our hospital for developmental delay at 36 months of age. He showed a facial dysmorphism, developmental delay and generalized seizures, which indicated a diagnosis of AS. Although the probability of recurrence of IC is up to 50%, which is the highest recurrence rate for PWS/AS, his younger sister, who is the second-born child, is healthy [25].

To the best of our knowledge, this study is the largest cohort covering both PWS and AS, with clinical information. A few previous studies dealt with the molecular diagnosis of these syndromes, but most of them focused only on PWS or presented no clinical findings [2, 26].

Our study has some limitations. First, there is an intrinsic limitation to MS-MLPA in that it cannot distinguish UPD from ID. To distinguish these two mechanisms, microsatellite analysis or single nucleotide variant analysis is required [27]. However, we successfully differentiated the IC deletion type, which has a 50% recurrence risk, using MS-MLPA. In addition, we could not evaluate the behavioral or psychological status of the patients due to the lack of such information in the medical records. Lastly, this was a retrospective and single-center study. Thus, a selection bias may exist, and further large-scale prospective studies for PWS and AS are needed.

AS caused by a UBE3A variant, which accounts for 20% of AS cases, cannot be diagnosed using MS-MLPA, and further diagnostic testing is needed. To date, only two patients have been diagnosed as having AS due to UBE3A pathogenic variants in our laboratory. Therefore, we propose the use of MS-MLPA as a first-line diagnostic tool for PWS or AS, in accordance with the EMQN/ACGS guidelines [28]. The next step would be UBE3A variant analysis of samples from patients who are highly suspected of having AS, as well as maternal samples.

In conclusion, MS-PCR and MS-MLPA show perfect diagnostic concordance, and MS-MLPA can substitute for MS-PCR. In addition, MS-MLPA provides more information about the molecular mechanisms underlying the diseases and may be a helpful tool for genetic counseling of families with PWS and AS. Finally, patients who are strongly suspected of having AS and show negative MS-MLPA results should undergo additional testing.