Prader–Willi syndrome (PWS, OMIM #176270) and Schaaf–Yang syndrome (SYS, OMIM #615547) are rare autosomal-dominant, imprinted genetic disorders caused by the loss of one or more normally active paternal genes in the chromosomal region of 15q11-q13, called the PWS region. The two main molecular causes of PWS include paternal 15q11-q13 deletion, which is present in 65–75% of individuals with PWS, and maternal uniparental disomy in which both chromosome 15s are from the mother, present in 20–30% of cases. The remaining individuals possess defects in the genomic imprinting center (IC) or chromosome 15 translocations or inversions
[1][2][3]. PWS affects approximately one in 15,000–20,000 individuals with around 400,000 cases worldwide
[4], while the prevalence of SYS is <1/1,000,000
[5]. Major clinical features of PWS include intellectual and physical disabilities, obesity, maladaptive behaviors, and several endocrine dysfunctions, like growth retardation and hypogonadism
[6]. SYS shares several symptoms with PWS yet is distinct. The underlying genetic cause of SYS is the disrupted expression of
MAGEL2, one of the protein-coding genes within the PWS region on chromosome 15, due to mutations in the paternal copy
[7][8][9].
2. PWS-Associated Genes, Their Imprinting, and Expression Pattern
2.1. MKRN3
MKRN3, a highly conserved PWS-associated gene sharing 82% similarity between humans and mice, is ubiquitously expressed in human and mouse tissues with high levels in the brain and testis
[10][11][12]. The hypothalamic
MKRN3 expression is high early in life and decreases before puberty initiation, an evolutionarily conserved pattern observed in mice, rats, and non-human primates
[13]. MKRN3 belongs to the Makorin family of proteins that contain two to four C3H zinc finger domains, a unique Cys-His configuration, and a RING zinc finger domain that is critical for the activity of the RING subfamily of E3 ubiquitin ligases
[12][14]. Paternal deletion or loss-of-function mutations in
MKRN3 are thought to contribute to hypogonadism, infertility, and the rare cases of central precocious puberty (CPP) in PWS patients
[10][15][16][17][18]. CPP is characterized by elevated expression and secretion of hypothalamic gonadotropin-releasing hormone (GnRH), resulting in the early development of secondary sexual characteristics
[15][19].
Mkrn3 knockout mice phenocopy many symptomatic features of human CPP
[20]. Although MKRN3’s function in regulating puberty initiation in mammals is not completely understood, CPP-associated mutations in
MKRN3 result from reduced expression or loss of E3 ubiquitin ligase function. MKRN3 ubiquitinates MBD3 (methyl-DNA binding protein 3) and epigenetically silences
GNRH1 [20]. Furthermore, MKRN3-mediated ubiquitination of poly(A)-binding proteins destabilizes
GNRH1 mRNA in the hypothalamus
[19]; thus, MKRN3 regulates GnRH on the transcriptional and translational level. MKRN3 also inhibits puberty onset through interaction with other proteins, including IGF2BP1 and NPTX-1
[21][22][23]. Additionally, an in vitro luciferase assay showed that MKRN3 inhibited the expression of kisspeptin and neurokinin B, neuropeptides that stimulate GnRH secretion
[13], though only neurokinin B protein levels were increased in
Mkrn3 knockout mice
[21]. In short, MKRN3 is a neuroendocrine inhibitor upstream of GnRH, and
MKRN3 loss-of-function mutations are the main genetic cause of CPP, including in PWS patients
[19][20][24][25].
2.2. MAGEL2
MAGEL2 is a member of the melanoma-antigen (MAGE) gene family, which expanded from one gene in lower eukaryotes to more than 40 genes in eutherian mammals
[26].
MAGEL2 encodes for a regulator protein of an E3 ubiquitin ligase and affects the retromer-dependent endosomal protein recycling
[7][27][28][29].
MAGEL2 is highly expressed in the central nervous system, especially in the hypothalamus and placenta
[7][30].
Magel2-null mice recapitulate many PWS features, like poor sucking and obesity
[31][32]. The loss of
MAGEL2 leads to decreased neuropeptide and hormone production and impaired hypothalamic secretion. Further details about
MAGEL2's function can be found here:
[33].
2.3. NECDIN
Necdin (encoded by
NDN) is another member of the MAGE family within the PWS region.
NDN is highly expressed in certain regions of the brain, such as the locus coeruleus and hypothalamus and placenta
[26][34][35][36]. In particular,
NDN is highly expressed in GnRH neurons in the mature hypothalamus
[37]. Necdin plays an integral role in neuronal differentiation
[35], so Necdin deficiency leads to widespread nervous system abnormalities
[38]. Deletion of
Ndn in mice recapitulates several PWS symptoms, including neonatal mortality, altered pain threshold, hypogonadism, and sensory–motor defects
[38][39][40], and significantly reduces the quantity of hypothalamic GnRH neurons
[36][41]. Muscatelli et al.
[42][43] reported a significant reduction in oxytocin-expressing neurons in the lateral parts of the paraventricular hypothalamic nucleus of
Ndn-deficient mice.
Necdin-deficient mice also exhibit disturbed migration of serotonin neuronal precursors and increased serotonin transporter activity that causes apnea, making
Ndn knockout mice the only model that reproduces the respiratory challenges of the PWS
[40][43][44]. Further, Necdin and Magel2 together were shown to control leptin receptor sorting and degradation through a ubiquitin-dependent pathway, including E3 ubiquitin ligase Rnf41, deubiquitinase Usp8, and protein Stam1, contributing to obesity in PWS
[32]. More recently, Necdin was reported to regulate the stability of BMAL1, one of the core transcription regulators of the circadian rhythm, potentially contributing to the disturbed circadian rhythm observed in patients
[34].
2.4. NPAP1
The imprinted
NPAP1 is a primate-specific gene encoding a nuclear pore complex-associated protein from a POM121-related family of retrogenes with testis-specific expression, a unique pattern among the PWS genes
[45][46][47]. Interestingly,
NPAP1 is the only PWS gene not conserved in rodents. Rather, the PWS syntenic region on chromosomes 7 and 1 in mice and rats, respectively, contains another coding gene,
Frat3, that is potentially involved in WNT signaling during embryonic development
[48].
2.5. SNURF/SNRPN
SNURF/SNRPN (SNRPN upstream reading frame (SNURF)/small nuclear ribonucleoprotein polypeptide N (SNRPN)) is a complex gene locus belonging to the SNRPN SmB/SmN family. The protein plays a role in pre-mRNA processing, tissue-specific alternative splicing events, and transcript production
[4]. The
SNURF/SNRPN gene is a bicistronic transcript that encodes two proteins and also contains the
snoRNA genes
[49][50][51][52]. Chromosomal deletions that affect the
SNRPN upstream exons and the imprinting center (IC) cause PWS by impairing the allele-specific expression of genes normally subject to the imprinting control
[53]. It has also been reported that even a single small deletion or single-nucleotide variant involving
SNURF/SNRPN causes major symptoms of PWS including hypotonia, dysmorphic features, intellectual disability, and obesity
[54][55].
2.6. SNORD116
The non-coding RNA molecule
SNORD116 is highly expressed in the brain
[56], and clinical evidence from rare patients with
SNORD116 deletions or translocations indicates that the
SNORD116 cluster is crucial for most of the PWS phenotypes
[10][57][58][59][60][61][62]. In mice, global or selective deletion of
Snord116 from hypothalamic neurons causes low birth weight, increased weight gain in early adulthood, increased energy expenditure, and hyperphagia
[63]. One group reported that
Snord116 paternal knockout (
Snord116m+/p−) mice also had reduced transcript levels of the prohormone convertase PC1 (encoded by
Pcsk1), impairing prohormone processing and possibly causing the major neuroendocrine features of PWS
[64]. Chen et al.
[65] also observed a reduction in PC1 protein levels in pancreatic islets from
Snord116m+/p− mice, although a follow-up study found no differences in the hypothalamic
Pcsk1 transcript levels in
Snord116m+/p− mice
[66].
Besides PWS and SYS, Angelman syndrome (AS, OMIM#105830) is another imprinting disorder that is caused by genetic variation in the same region of chromosome 15. Ataxia, happy demeanor, and sleeplessness are some of the symptoms observed in individuals with AS
[67][68]. In AS, the maternal copy of the genes in 15q11-q13 is missing, while the paternal copy is inactivated in PWS and SYS
[7][68]. The PWS region is maternally imprinted, and genes must be expressed from the paternal chromosome. In contrast, the adjacent AS region is paternally imprinted, and encoded genes must be expressed from the maternal chromosome.
2.7. Genomic Imprinting
Genomic imprinting in PWS and AS causes a monoallelic expression of genes and is regulated by a bipartite IC, composed of the PWS-IC and AS-IC, that establishes local imprinting regulation of multiple genes within the 15q11-q13 region
[69][70]. The PWS-IC comprises a CpG island and is associated with the 5′ flanking region, the first exon and 5′ end of the first intron of
SNRPN [71]. The AS-IC is located 35 kb upstream of the
SNRPN promoter
[72]. The PWS-IC is differentially methylated on the maternal allele, with the paternal allele remaining in an open, unmethylated state
[51][70][73][74][75]. Interestingly, deletion of AS-IC on the maternal allele also leads to the biallelic unmethylation of neighboring PWS-IC, suggesting that AS-IC contributes to establishing the methylation state and closed chromatin structure of PWS-IC
[70][76]. Paternally inherited deletion of the PWS-IC results in loss of expression of
MAGEL2 and other PWS genes
[3][71]. For example, upon deletion of murine paternal PWS-IC, Brant et al.
[77] noted a two-fold increase in the methylation level of CpG sites at differentially methylated regions (DMRs) of paternally expressed PWS genes, including
Magel2, while deletion of maternal PWS-IC resulted in no methylation changes.
Although there are contradictory findings regarding when the maternal PWS-IC becomes methylated, in oocytes or post-fertilization, most results suggest that methylation occurs after the blastula stage
[78][79][80][81]. A proposed model for PWS imprinting regulation suggests that methyl groups are removed from PWS-IC during both spermatogenesis and oogenesis, and then AS-IC interacts with PWS-IC to facilitate de novo methylation of the maternal PWS-IC after fertilization
[71]. After fertilization and establishment of methylation of the maternal PWS-IC, the unmethylated paternal PWS-IC functions as a promoter for the
SNRPN transcription unit and acts at long distances to activate transcription of
MAGEL2, amongst others
[71]. However, the exact mechanism and proteins involved in this intricate process are unclear.
2.8. Expression Pattern
In addition to the epigenetic regulation of imprinted genes in the PWS region, the distinct tissue expression patterns of these genes clearly suggest specific transcriptional regulation; however, the details of potential transcription factors involved are almost completely unknown. Interestingly,
MAGEL2 and
NDN are expressed at the highest levels in the brain (especially
MAGEL2 in the hypothalamus), pituitary, and placenta
[30][82], tissues linked to the evolution of genomic imprinting in eutherian mammals. For instance, most of the imprinted genes (~230 genes known so far) are expressed in the placenta and several now have well-known roles in placental biology, fetal growth, and homeostasis of pregnancy
[83][84]. The functional convergence of many imprinted genes on the placenta is in line with the hypothesis that genomic imprinting evolved in mammals because of the conflicting interests of maternal and paternal genes in relation to the transfer of nutrients from the mother to her offspring
[85][86]. Very little is known about the role of the PWS genes in the placenta and warrants future investigation.
Furthermore, genomic imprinting is increasingly appreciated for its role in the nervous system. Outside of the placenta, the brain is one of the adult tissues with the largest number of expressed imprinted genes
[87][88]. In a recent tissue expression analysis on a single-cell level, imprinted genes were found over-represented in murine hypothalamic neurons
[89]. These data support the previously suggested role of imprinted genes in the neuroendocrine hypothalamic regulation of diverse physiological functions
[90]. Indeed, the first insights into the physiology of imprinted gene function were derived from studying neuroendocrine symptoms in PWS patients and animal models
[91]. Through connections with the pituitary, the hypothalamus regulates hormone release in distal endocrine glands, including adrenals, thyroid, and gonads, to control key physiological processes, like stress response, growth, and reproduction. In addition, the hypothalamus makes neural connections via the autonomic nervous system and other pathways to regulate sleep, body temperature, and feeding
[92], several of which are disturbed in PWS and SYS (
Figure 1).
Figure 1. Comparison of PWS and SYS phenotypes in humans (A) and rodent models (B).
To summarize, mouse models and unique genetic backgrounds of patients revealed how single or multiple genes contribute to the development of PWS and SYS symptoms. The unique expression pattern of PWS genes (e.g., MAGEL2), their evolutionary appearance, and their molecular and cellular roles suggest that some of the PWS genes evolved in mammals as tissue-specific regulators of hypothalamic neuroendocrine function.