Study of SNRPN genetic and epigenetic mutations in Prader-Willi and Angelman patients

S. Yu. Chernushyn, N. V. Hryshchenko © 2018 S. Yu. Chernushyn et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 606:61+575.224.2+577.218+612.821.5+616-092


Introduction
Prader-Willi syndrome (PWS) is characterized by hypotonia, failure to thrive with poor suck, hypogonadism, short stature with small hands and feet, hyperphagia leading to morbid obesity, beginning from early childhood; developmental delay/intellectual disability, and behavioral issues, including obsessive compul-sive disorder. The characteristic facial features are also evident. Angelman syndrome (AS) is characterized by developmental delay, intellectual disability, absent speech, seizures, ataxic gait, easily excitable happy demeanor, and characteristic faces.
PWS and AS typically result from the deletion of paternal for PWS or maternal for AS copies of the chromosome region 15q11-q13. ISSN 1993-6842 (on- Each one occurs with a frequency of approximately 1/15,000 to 1/30,000 live births [1], in the vast majority of cases they happen de novo, in families with no history of these syndromes. Chromosome 15q11-q13 is a region that harbors several genes regulated by genomic imprinting, a phenomenon when genes are expressed preferentially from one parental allele. As a result, the genes subjected to regulation by genomic imprinting are functionally haploid, having only a single functional copy [2]. At least two genes SNRPN and NDN from the 15q11-q13 region have differentially methylated CpG islands in their promoter regions that are methylated on the maternal chromosome leading to the silencing of the maternal allele. The alteration of the paternal copy of the imprinted SNRPN gene will cause PWS [3], and vice versa, the loss of maternal allele leads to AS. The other gene in this region, encoding the ubiquitin-protein ligase E3A -UBE3A, is a parentally imprinted gene in which the paternal allele is selectively silenced in mature neurons. The promoter of UBE3A gene is completely unmethylated and it is active on both parental copies in all tissues, silencing of the paternal allele expression is achieved by noncoding antisense transcript UBE3A-ATS [4], which is transcribed backwards to the UBE3A.

Biomedicine
There are 4 known genetic mechanisms which can cause PWS and AS: de novo deletions involving the chromosome 15q11.2-q13 region, uniparental disomy (UPD) of 15q11.2-q13, imprinting center (IC) defects and point mutations in the causative gene (Table 1). About 20 % of cases of AS are caused by mutations in the UBE3A [5] but there are no instances of a point mutation in any gene caus-ing PWS, suggesting that PWS is a true contiguous gene syndrome, resulting from the loss of more than one gene. The deletion of the 15q11.2-q13 locus occurs at a frequency of about 1/15,000 newborns and is probably one of the most common pathogenic deletions observed in humans.
For molecular genetic diagnostics of PWS and AS, the most commonly used method is the analysis of the SNRPN gene using FISH [7]. This method allows identifying only deletions in the studied region. Therefore, the use of methods capable of detecting UPDs and methylation defects significantly increase the informativeness of the molecular diagnosis of these syndromes.
The aim of the study was an estimation of 15q11.2-q13 rearrangements and epigenetic alterations frequency in the group of Ukrainian patients with PWS and AS clinical phenotype. The analysis of the SNRPN gene alterations by methyl-spesific PCR was chosen because it allowed simultaneous testing of three types of alterations: deletions, UPDs and IC defects.

Methods
Group of 60 Ukrainian patients with PWS phenotype (18 females and 42 males) and 32 patients with AS phenotype (21 and 11 respec-tively) were referred from the regional medical genetics centers of Ukraine to the IMBG from 2011 to the present. The informed consent for clinical and genetic study was obtained from the patients statutory trustees in compliance with national ethics regulation.
DNA was extracted from the patients blood samples using proteinase K cell lysis and standard phenol-chloroform DNA purification. To identify methylation status of the SNRPN gene all collected samples were analyzed by methylation-specific PCR (MS-PCR). Bisulfide (BS) conversion of genomic DNA was carried out using EZ DNA Methylation-GOLD TM Kit (Zymo Research) according to the manufacturer's protocol. We used 1-2 μg of genomic DNA for BS-conversion, the converted DNA was diluted in 10-15 μl of the elution buffer. To find out the methylation status of the 15q11-q13 locus we used PCR with two primers pairs: one specific to the SNRPN gene BSconverted methylated DNA, second -to the SNRPN gene BS-converted unmethylated DNA. PCR was carried out in a total volume of 20 μl containing 1x FIREPol® Master Mix with 2.5 mM MgCl 2 (Solis BioDyne), 0.75μM primers (both for the methylated and unmethylated BS-converted SNRPN gene fragments) and 10μl of BS-converted DNA solution. The PCR conditions were 1 cycle -95°C for 5 min followed by 35 cycles of the 3 step protocol: 95°C -20 sec, 62°C -30 sec, 72°C -30 sec. The MS-PCR results were visualized by 1.5 % agarose gel electrophoresis and ethidium bromide staining.

Results
The MS-PCR technique used in this work allows the differential detection of both types of normal alleles of the SNPRP gene (methylated and unmethylated). The presence of two fragments (174 and 100 bp) corresponds to the presence of two normal homologues of the 15th chromosome: methylated, inherited from the mother (174 bp) and unmethylated -from the father (100 bp) ( fig. 1). Thus, it is possible to reveal the presence of a deletion in methylated or unmethylated allele, a uniparent disomy (the presence of only 2 methylated or only 2 unmethylated alleles) and mutations in IC, leading to the absence of one or another type of allele. Based on the literature data presented in Table 1, PWS can occur only as a result of 3 types of alterations of the 15q11.2 chromosomal region: paternal deletions, maternal uniparent disomy or mutations in the IC. So, the analysis of the SRNPN gene by MS-PCR could detect about 100 % of the alterations associated with PWS. Considering that in a third of cases AS is caused by the UBE3A mutations, the informativeness of the MS-PCR for this disease does not exceed 70 %. Additionally, one of the disadvantages is that this method, identifying 3 types of damage, does not allow their differentiation. In the AS group the frequency of SNRPN methylated allele loss was detected in 28 % (9 of 32) of patients. There are also 3 possible genetic causes -maternal deletions/paternal UPDs/IC, but the most likely one, based on literary data, is the deletion in the 15q11.2 of maternal origin.

Discussion
The deletions of the chromosome 15q11.2-q13 region are typically diagnosed by fluorescence in situ hybridization (FISH) using the SNRPN probe [7]. With this method, it is possible to confirm the diagnosis in 60-70 % of PWS and AS patients, but not those caused by UPDs or IC defects.
Based on the results obtained, it can be argued that the application of the MS-PCR technique to verify PWS is the most informative and low-cost in comparison with the FISH. However, in our study, close to 100 % confirmation of PWS was not obtained. Lower than expected detection rate in the PWS group can be explained by the presence of patients with PWS-like syndromes. We can assume that proximal spinal muscular atrophy type I (SMA I, OMIM # 253300) may be one of the probable diseases in children younger than 18 months when PWS was not confirmed. Both of these diseases SMA I and PWS are characterized by generalized hypotonia at the age of 12-18 months [8][9]. In case of PWS, these symptoms do not progress and are fade away when a patient reached the second year of life, in contrast to SMA I. This may be confirmed by the fact that in one patient with suspected PWS, we identified homozygous deletions in the SMN1 gene, which are major mutations in SMA I. Therefore, it is possible to recommend additional clinical and genetic studies to the patients under 18 months, who have idiopathic hypotonia, for differentiation of the diagnosis. Among clinical studies, the most informative, in our opinion, may be electromyography, which allows detecting muscular and neural alterations that lead to muscular or neuronal hypotonia and are not associated with PWS.
Moreover, in the PWS group we observed the significant prevalence of males -70 % (42 of 60), but the frequency of detected alterations in females was higher than in males -56 % (10 of 18 patients) and 36 % (15 of 42) correspondently. This can indicate that less strict diagnostic criteria for males lead to incorrect over diagnostics of PWS in the current group.
The low detection rate in AS (28 %) could be due to both clinical misdiagnosis and limitations of the used method of molecular ana lysis. To improve the molecular diagnostics of AS, the further analysis of the AS associated genes, such as the UBE3A and MECP2 genes should be done. We suggested that in addition to the mutations in the UBE3A gene, the mutations in the MECP2 gene can lead to the AS phenotype. This assumption is based on the literature data about the similarity of the clinical phenotype in the patients with Rett syndrome and AS [10,11].

Conclusions
Analysis of the SRNPN gene region by MS-PCR is cost-effective and high informative, therefore it could be implemented for the PWS and AS molecular diagnostics, rather than FISH. Our results revealed that the proposed molecular test can exclude the clinical diagnosis of PWS in approximately 60 % of Ukrainian patients with suspected PWS. In 30 % of the suspected AS patients, this analysis confirms the clinical diagnosis. Our nearterm plans include implementation of further molecular analysis of the AS associated genes, such as the UBE3A and MECP2 genes, to improve the detection rate of molecular diagnostics of AS.