a more detailed look...
Introduction
Prader-Willi syndrome (PWS) is a neural developmental disorder that is characterized by infantile hypotonia, feeding difficulties, hypogonadism, mental deficiency, hyperphagia/obesity, learning problems, and behavioral difficulties [2]. The genes are differently imprinted on maternal and paternal chromosomes, so both copies are needed for normal development. PWS can occur due to a deletion in paternal chromosome 15q11-q13, maternal uniparental disomy (UPD) 15, or mutations involving the imprinting center [2]. Deletions account for about 70% of patients; 25% due to UPD and 2 to 5% for abnormalities in chromosome 15 [2]. In terms of significance, PWS has an incidence of about 1 in 15000 live births [6]. This review will discuss the etiology, diagnosis, and treatment of PWS.
Etiology
The chromosome 15q11-q13 region consists of 5 groups of important genes. They are the nonimprinted genes, paternally expressed genes, maternally expressed genes, genes with paternal biased expression, and gene whose expression status is unknown. The nonimprinted genes include GCP5, CYFIP1, NIPA2, NIPA1, C15ORF12, and P (OCA2). Paternally expressed genes (expressed only on the paternal chromosome 15) contain MKRN3, MAGEL2, NDN, SNURF-SNRPN, and the snoRNAs genes. The maternally expressed genes, the genes silent on paternal chromosome 15, are UBE3A and ATP10C. GABRB3 and GABRA5 are genes with paternal biased expression; and GABRG3 is the one gene whose expression status is not confirmed [2; 12].
Deletions can be divided into two groups, type I and type II. The proximal breakpoint (BP) in the 15q11-q13 region occurs at 1 of 2 sites. The larger type I deletion is from BP1 to BP3, which involves all genes mentioned above [2; 12]. The smaller type II deletion is from BP2 to BP3 and doesn’t involve NIPA1, NIPA2, CYFIP1, and GCDS [2; 12]. For maternal UPD 15, the main mechanism thought to be responsible is trisomic rescue of trisomic conception with the loss of paternal chromosome 15 [6]. Individuals with maternal UPD lack paternal chromosome 15 and expression of the associated genes. These changes in gene composition can lead to PWS.
The imprinting of the PWS domain is regulated through a bipartite cis-acting IC, comprised of PWS-IC and AS-IC, within and upstream of the SNRPN promoter in the 15q11-q13 region. The paternal allele is unmethylated and actively transcribed and the maternal allele is methylated and silent; along with other processes, this leads to imprinting [3]. Wu et al shows that mice without two Rb-binding protein-related genes, Rbbp1/Arid4a and Rbbp1l1/Arid4b, go through abnormal modifications at the PWS-IC [3]. There was a reduced trimethylation of histone H4K20 and H3K9 and reduced DNA methylation, changing the maternal allele toward a more paternal epigenotype. In addition, mutations of Rbbp1/Arid4a, rbbp1l1/Arid4b, or Rb suppressed an AS imprinting defect [3]. These results have identified some regulators, but more research is needed to confirm whether mutations in Arid4a and Arid4b can contribute to the epigenetic mechanisms of the disease [3].
In one study, Bittel et al did a semiquantitative analysis of expression of the genes/transcripts located in or close to the PWS critical region [5]. Microarray analysis was done on lymphoblastoid cells from nine young adult males with either deletions, UPD, or no mutations (control). No expressions of paternal genes in the deletion or UPD cell lines were detected [5]. All cell lines showed similarity in expression in genes located outside the 15q11-q13 region [5]. In addition, there were no differences in the expression levels of biallelically expressed genes from within 15q11-q13 when compared UPD with controls [5]. In contrast, two maternally expressed genes, UBE3A and ATP10C, were more highly expressed in UPD cell lines than controls and deletion [5]. Interestingly, several genes/transcripts, like GABRA5 and GABRB3, had increased expression in UPD than in deletion cell lines, but were still lower than controls [5]. While it is still not clear how these genes interact, these differences in expression may contribute to the phenotype of the disease.
Hyperphagia (increased food intake) is a major symptom of PWS. Holsen et al used functional magnetic resonance imaging (fMRI) to study neural mechanisms underlying food responses [4]. In the healthy weight control (HWC) group, the response to food pictures in the amygdale, orbitofrontal cortex, medial prefrontal cortex (PFC), insula, hippocampus, and parahippocampal gyrus were more activated before eating than after [4]. For the PWS group, the activation to food remained high after meal [4]. These data demonstrated that the brain is abnormal in the PWS group.
The lack of expression of genes like NIPA1, NIPA2, and CYFIP1 are thought to have an impact on nervous system development and/or function. NIPA1 was reported to encode a Mg2+ transporter as immunoblot and immunofluorescence demonstrated its localization in early endosomes and the plasma membrane, and their redistribution in response to magnesium flux [11]. NIPA2 is conserved in vertebrates and widely expressed, including in CNS. Like NIPA1, cDNA analysis indicated that it encodes putative polypeptides with nine transmembrane domains, suggesting receptor or transporter function [12]. CYFIP1 was shown, using a yeast two-hybrid system, to interact with FMRP, whose absence causes Fragile X Syndrome. Previous studies also identified CYFIP1 as an interactor of the Rac1 small GTPase. It’s a possibility that CYFIP1 and Rac1 are regulators of FMRP [13]. Data from these studies suggest that NIPA1, NIPA2, CYFIP1 may have a great influence on the studied behavioral and cognitive parameter. Unfortunately, additional research is needed to fully understand the influence of these gene expressions on PWS.
NDN, MAGEL2, and HBII-52 are paternally expressed genes. NDN, or necdin, was shown to interact with the cell cycle-regulation factor E2F1 using chromatin immunoprecipitation analysis [14]. Other methods like TUNEL staining and PCR informed its role in reducing neuronal apoptosis in cerebellar granule neurons, by suppressing the E2F1-Cdc2 system [14]. Therefore, unexpression of NDN would likely lead to neurodevelopmental disorder seen in PWS. Visualized by LacZ histochemistry and immunohistochemistry, Magel2 transcripts are seen highly enriched in the suprachiasmatic nucleus, playing a role in the circadian output pathway [15]. Along with reduced food intake, knockout mice (Magel2m+/p-) ran significantly less, and in more frequent and shorter bouts [15]. HBII-52 regulates alternative splicing of 5-HT22R by binding to a silencing element in exon Vb, analyzed by RT-PCR [16]. These results have only suggested possible gene functions and have yet studied all the pathways leading to PWS.
Diagnosis
The symptoms for PWS are severe hypotonia, feeding difficulties, hypogonadism, small hands and feet, developmental delay, behavior problems, and genital hypoplasia [2]. Children with PWS between 2 to 4 years old tend to have an avid appetite, which resulted in obesity if food intake is not regulated [2]. Indeed, Prader-Willi syndrome is the most common genetic disorder related to human obesity. It was reported that ghrelin, a potent orexigenic and adipogenic hormone, is significantly elevated in patients with PWS. Ghrelin can increase food intake, gastric acid secretion and motility, and fat deposition [9]. However, it is still unknown how the behavioral disorders result from the lack of expression of imprinted genes in the paternal 15 chromosome.
Hypopigmentation, homogeneous, and higher visual processing of complex stimuli were more often seen in individuals with PWS with a deletion than those with UPD. On the contrary, higher verbal IQ scores, fewer maladaptive behaviors, and reduced self-injury are seen associated with UPD individuals compared to those with deletions [2]. In addition, Bittel et al had previously presented that persons with type I deletions had more behavioral and psychological problems than those with type II deletion or UPD. These two regions of deletion break points are defined by microsatellites [6]. Not all PWS individuals are created equal and the lack of gene expressions determines the severity of the disease.
There are several ways for diagnosing PWS based on genetics. Some deletions can be detected by cytogenetic techniques, but most are detected by molecular assays [6]. Methylation can detect deletions, UPD, and mutations, but cannot distinguish among them so cytogenetic and microsatellite analyses are used to confirm. These detection methods use broad-slot gels and simultaneous hybridization of a non-chromosome 15 constant marker that is detected by molecular probes. Either cytogenetic or molecular means were able to determine if the deletion is paternal [1; 6]. In addition, PWS patients with UPD are found to be heterozygous and identical to their mother, using probes p189-1 and p3-21 [1]. Borelina et al used a variety of cytogenetic and molecular approaches like chromosome G banding, fluorescent in situ hybridization, and a DNA methylation test [6].
PWS can also be physically measured, since PWS patients have abnormal physiological and brain functions. For example, Shu et al measured blood pressure, weight and height using a super-view H-101 meter, and body composition by an InBody 3.0 analyzer. In addition, Wechsler Intelligence Scale in Children and Wechsler Adult Intelligence Scale were performed by experienced psychologists on PWS patients; the average IQ score was 52 with a range of 40-71 [7]. Another method involving diffusion tensor imaging, based on a high-field MRI system, was used to detect brain developmental abnormalities in PWS patients [8]. PWS patients had higher trace value but lower fractional anisotropy than normal controls [8].
Treatment
Even though there is no definite cure, two treatments for PWS are discussed as having great impact. One involves the use of growth hormone. It takes into account the decline in growth hormone (GH) secretion in PWS to use GH administration as treatment. Children with PWS, undergoing GH therapy, experienced improved body composition with daily doses of at least 1 mg/m² [10]. Obesity, poor growth, and hypotonia in children with PWS are accompanied by abnormal body composition, resembling a GH-deficient state. It was known that hypothalamic dysfunction in PWS includes decreased GH secretion. Aaron et al showed that salutary and sustained GH-induced changes in growth, body composition, bone mineral density, and physical function in children with PWS can be achieved with daily administration of GH doses ≥ 1 mg/m ². Lower doses are not as effective. They tested 46 children with PWS, who had had GH therapy for 12-24 months. The percent body fat, lean muscle mass, and bone mineral density were measured by dual x-ray absorptiometry. In addition, indirect calorimetry was used to determine resting energy expenditure and to calculate respiratory quotient; and a Bruininks-Oseretski test of physical performance was used to evaluate strength and agility [10]. These studies show that GH can play a possible therapeutic role in PWS patients.
The other treatment uses Exendin (Ex)-4, a peptide isolated from the venom of the lizard Heloderma Suspectum. Ex-4 is still in clinical trial, but its result is promising. It can reduce obesity by decreasing ghrelin levels up to 74% [9]. Ex-4 is an agonist of the GLP-1 receptor. It has the same effects as Glucagon-like peptide (GLP)-1, which can reduce food intake, weight gain, and fat deposition in obese rats; it has potent insulinotropic properties in rats and humans. One big difference is it can resist DPP-IV, which rapidly inactivates GLP-1. In one experiment, Ex-4 was found to reduce ghrelin level up to 74% reduction in fasted rats. The decreased in ghrelin reduced food intake in fasted rats and the effects were dose dependent and long lasting (up to 8 h). Also, Ex-4 was independent of the levels of leptin and insulin. Rats were intracerebroventricularly and intraperitoneally administered the drugs, and total ghrelin level was measured using a radioimmunoassay (RIA). These results show that Ex-4 can be used as a therapeutic treatment for PWS patients with high amount of ghrelin [9].
Conclusion
Prader-Willi syndrome is caused by the lack of expression of genes on paternal chromosome 15. It is unknown how these genes interact, but differences in their expression lead to the phenotype of the disease. Therefore, it is best we understand the underlying mechanism through more research, which would help us develop a definite cure. For now, there are two potential treatments: growth hormone, in use, can improve body composition, while Ex-4 was shown to reduce obesity in rats. PWS patients can try these treatments and hope for something better to come along, soon.
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