Clinical Application of an Innovative Multiplex-Fluorescent-Labeled STRs Assay for Prader-Willi Syndrome and Angelman Syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by absence of paternally or maternally expressed imprinted genes on chr15q11.2-q13.3. Three mechanisms are known to be involved in the pathogenesis: microdeletions, uniparental disomy (UPD) and imprinting defects. Both disorders are difficult to be definitely diagnosed at early age if no available molecular cytogenetic tests. In this study, we identified 5 AS patients with the maternal deletion and 26 PWS patients with paternal deletion on chr15q11-q13 by using an innovative multiplex-fluorescent-labeled short tandem repeats (STRs) assay based on linkage analysis, and validated by the methylation-specific PCR and array comparative genomic hybridization techniques. More interesting, one of these PWS patients was confirmed as maternal uniparental isodisomy by the STR linkage analysis. The phenotypic and genotypic characteristics of these individuals were also presented. Our results indicate that the new linkage analysis is much faster and easier for large-scale screening deletion and uniparental disomy, thus providing a valuable method for early diagnosis of PWS/AS patients, which is critical for genetic diagnosis, management and improvement of prognosis.


Introduction
Prader-willi syndrome (PWS) and Angelman syndrome (AS) are the first known human genomic imprinting disorders, each with an estimated prevalence of 1/15000-1/25000 in live births. PWS and AS were initially identified by high-resolution chromosome banding, in which patients were found with an interstitial deletion in the same region of chromosome 15q11.2-q13.3 [1]. There is ample evidence that a cluster of genes located in the chromosome region 15q11q13 were expressed from solely paternal or maternal chromosome. It was considered that the deficiency of UBE3A gene from the maternal chromosome could lead to AS [1,2]. While the loss of function mutation in SNORD116 snoRNAs could cause the key characteristics of the PWS phenotype [1,3].
PWS patient presents particular manifestations, such as hypotonia, poor suck and feeding difficulties in infancy, then is accompanied by hyperphagia, hypogonadism, hypopigmentation, short stature, cognitive impairments and distinct facial features [4,5]; while AS is characterized by microcephaly, severe intellectual disability, gait ataxia, seizures, an apparent happy demeanor including inappropriate laughter and excitability [6].
Both disorders have clinically overlapped features with other diseases during infancy, which makes them difficult to diagnose solely relying on the clinical manifestations. The appearance of hyperphagia and morbid obesity provide useful information for clinical diagnosis of PWS; while the distinctive behavior of apparent happy demeanor and seizures are clinically suspicious of AS. The definite diagnosis of PWS/AS, however, is much dependent on genetic tests. Currently, there are some techniques to identify the disorders, such as G-banding karyotyping, fluorescence in situ hybridization (FISH), methylation-specific PCR (MS-PCR), multiplex ligation-dependent probe amplification (MLPA), short tandem repeat (STR) linkage analysis, DNA sequencing, array comparative genomic hybridization (array CGH) and chromosomal microarray analysis (CMA) [8,9].
In this study, we establish an innovative STR linkage analysis based on multiplx-fluorescentlabeled PCR to detect the AS/PWS patients and then validated by MS-PCR and array CGH. Five AS patients with the maternal deletion and 26 PWS patients with paternal deletion at 15q11-q13 in Chinese population were identified One of the PWS patients was diagnosed as maternal uniparental isodisomy. The clinical characteristics of these individuals were also included.

Ethics statement
The work was approved by Ethics Committee of Qilu Children's Hospital of Shandong University and Ethics Committee of Guangdong Women and Children's Hospital of Guangzhou Medical University. The individuals in this manuscript have given written informed consents (as outlined in PLOS consent form) to publish these cases details. The patients' information was anonymized prior to submission. All the procedures performed in the study were in accordance with the Declaration of Helsinki.
Qilu Children's Hospital of Shandong University as well as Guangdong Women and children's Hospital. The diagnosis of PWS and AS was made based on the clinical manifestations and genetic tests. The demographic characteristics and major clinical features of 31 patients were summarized in Table 1.
Peripheral blood samples were collected from the patients and their parents. Genomic DNA was extracted using TIANamp Blood Genomic DNA Purification Kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions.

STR linkage analysis
The STR markers were selected according to their high heterozygosity and location in the typical deletion region (BP1 to BP3) and the adjacent region of PWS and AS. The selected seven STR markers; D15S11, D15S646, D15S817, D15S128, D15S1513, GABRB3, D15S822 were located in the typical PWS and AS deletion region of BP1 to BP3; while additional two loci of D15S659 and FES were in the distal region near telomere. The reference sequence was obtained from the UCSC Genome Browser website (http://genome.ucsc.edu/index.html). The distribution of these loci was shown in Fig 1. Three multiplex-fluorescent-labeled polymerase chain reactions (PCR A, B and C) were set up with three forward primers labeled different fluoresceins of FAM, JOE and TAMRA in each PCR mixture of A, B and C. The multiplex PCR A was used to identify D15S659 (A1), D15S817 (A2) and D15S1513 (A3). The details of these loci and multiplexes were shown in Tables 2 and 3. The multiplex-fluorescent-labeled PCR A, B and C for all the 9 STR loci was optimized and performed for all the samples from the patients and their parents. The total volume of multiplex-PCR reaction was 50 μL and the reaction condition included: an initial denaturation at 94°C for 5 min, followed by 26 cycles of denaturation at 94°C for 30s, annealing at 61°C for 30s, and extension at 72°C for 30s, plus a final extension at 72°C for 20min. The PCR products were analyzed by capillary electrophoresis on an ABI 3100 sequencer (Applied Biosystems, CA,USA) in the presence of GeneScan 500 LIZ dye Size Standard and formamide (Applied Biosystems, CA, USA). GeneMapper software (Applied Biosystems) was used to provide DNA sizing and quality allele calls.

Methylation-specific PCR analysis
Methylation-specific PCR was performed according to the methods described by Liu et al [10]. The DNA was modified by sodium bisulfite (Takara, Dalian, China), which converts cytosine to uracil except for methylated cytosine that kept unchanged due to being resistant to bisulphate. The imprinting gene SNRPN containing a potential imprinting center for a chromosome domain on chr15q11-13, has been indicated that almost all CpG dinucleotides are methylated on the maternal chromosome but unmethylated on the paternal chromosome. The primers for the paternal-specific (unmethylated) and the maternal-specific (methylated) were shown in Table 3. The PCR was carried out in a 25μL reaction solution and the reaction condition included the initial denaturation at 94°C for 5min, followed by 40 cycles of denaturation at 94°C for 30s, annealing at 58°C for 30s, and extension at 72°C for 30s; and a final extension at 72°C for 7min. The PCR amplification products were visualized by a 3% agarose gel.

Array CGH
Eleven patients (case No: 1,2,3,5,9,25-30) went for the array CGH to detect the chromosomal breakpoints and validate the results of STR linkage analysis. The Agilent Human CGH Microarray 2 x 400K (Agilent, Santa Clara, CA, USA) was utilized following manufacture's instructions. Data were extracted using Feature Extraction software and analyzed using Agilent

STR linkage analysis
The STR linkage analysis was performed for all the samples from the patients and their parents. Five patients showed the maternal region deletion at the chromosome 15q11-q13 and were confirmed as AS; 26 patients demonstrated the paternal deletion in this region who were diagnosed as PWS. Of them, a 4-year-old girl patient from the family 6 (case 6) displayed two chromosome 15 alleles inherited only from one maternal chromosome 15 that was known as complete uniparental isodisomy of chromosomes 15, thus the patient was eventually identified as PWS with mUPD type. The results of all the patients with AS/PWS tested by STR linkage analysis were listed in Table 1 and S1

Methylation-specific PCR
The MS-PCR was carried out to validate the STR results. The products of 174 bp and 100 bp were obtained from methylated and unmethylated alleles of SNRPN gene locus. Five AS patients demonstrated the maternal region deletion at the chromosome 15q11-q13 (with only 100 bp of paternal fragment), while 26 PWS patients showed the paternal deletion in this  region (with only 174 bp of maternal fragment). The results were completely in conformity with the STR linkage analysis, thus clearly confirmed that these patients had PWS or AS ( Fig  3). However, it could not distinguish whether these PWS/AS patients were caused by deletion, UPD, or other chromosomal abnormalities without the STR linkage analysis.

Array CGH test
Array CGH test was performed for 11 cases, including 4 AS patients and 7 PWS patients. The results showed that 4 patients with AS had the identical 4.94Mb deletion at 15q11.2-q13.1.  However, 7 patients with PWS demonstrated different deletion fragments (S1 Table). These data were well consistent with STR linkage analysis though the deleted fragments were longer than that of STR results, demonstrating the reliability of this innovative STR linkage analysis for identifying PWS and AS.

Clinical features
Five AS and 26 PWS patients were diagnosed according to the diagnostic criteria described previously [11,12]. In the AS group, all the patients (

Discussion
Prader-willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by lack of paternally or maternally expressed imprinted genes at 15q11.2-q13.3, the PWS/AS critical region [1][2][3]. Three mechanisms are known to be involved in the pathogenesis: microdeletions, UPD and imprinting defects. The genetic tests are indispensable to identify the disorders and there have been some available techniques. For instance, G-banding karyotyping and FISH are traditional methods to detect the large deletions of chromosome. MS-PCR is currently the rapid and convenient test and can accurately identify 99% of PWS and 80% of AS individuals with deletion, UPD, and imprinting defects, but fail to differentiate from one another [2,10,13]. With the specialties of high accuracy, MLPA is known as the "gold standard' for detection of deletion but has a limitation of inability to differentiate between UPD and imprinting defects [13]. Chromosomal Microarray Analysis (CMA) technology including array CGH with characteristics of high resolution and high throughput has been widely used to detect copy number variants, including UPD in recent years. The sophisticated technology is more propitious to detect isodisomy, but not to heterodisomy. It has been reported that heterodisomy has high occurrence than isodisomy in PWS and AS, even other imprinting diseases [7]. Therefore, STR analysis are required to detect both deletion and UPD status. STR linkage analysis is a valuable tool to detect the gene location in chromosome with the capability of not only detecting deletion and UPD, but discriminate UPD status [10]. The STR linkage analysis based on direct sequencing could differentiate the molecular defect between typical deletion and UPD, exactly uniparental heterodisomy. However, the STR direct sequencing failed to make a distinction between both types of the deletion and uniparental isodisomy, as no differences between the sequencing results of the deletion and uniparental isodisomy [14]. The STR linkage analysis based on multiplex fluorescent-labeled PCR assay has the advantage over previous sequencing-based STR analysis. The significant and visualized differences of DNA quantity can be observed in the results (S1 Fig). Moreover, the multiplex-fluorescentlabeled STRs assay with only 5 hours, is more simple, rapid, and convenient in comparison with those tedious, time-consuming methods, such as MS-PCR experiment period for 2 days, MLPA for 2 days, STR linkage analysis based on direct sequencing for 3 days and array CGH for 5 days. In addition, the result of the STR linkage analysis is more intuitive. Our data indicated that the multiplex-fluorescent-labeled STRs assay based linkage analysis is an accurate, economic and practical method, not only applicable to identify the PWS/AS, but also diagnose and differentiate almost all imprinting disorders.
UPD is defined as a condition in which both homologous of a pair of chromosomal regions/ segments are inherited from only one parent, including isodisomy and heterodisomy. Isodisomy is referred to the inheritance of two copies from a single parental homologue, and heterodisomy is the inheritance of both homologues from one parent [15]. UPD can restore euploidy from a nondisjunction meiotic error to rescue the cell, but it carries two main types of developmental risks: the occurrences of the recessive inheritance diseases and the imprinting diseases. It has been reported that the incidence of UPD was about 1:3500 live births, 2-5% in AS and 25% in PWS [16,17]. Prader-Willi syndrome is the first known human genomic imprinting disorder, and mUPD of chromosome 15 accounts for 20%-25% in PWS. Complete isodisomy is possibly caused by monosomy rescue, post-fertilization error, or gamete complementation, which there is no reports about the incidence of PWS caused only by isodisomy [15]. Some researches had found that PWS with mUPD was less likely to be hyperphagia and excessive weight gain compared to those with deletion. The subtle phenotypic differences between the deletion and UPD genotypes might also be related to age at diagnosis [18].
In the study, all patients including 5 AS and 26 PWS were diagnosed based on clinical features and genetic analysis. For AS patients, the clinical features of seizures and happy disposition were remarkable in the infant cohort thus the diagnosis was made earlier than that for PWS. The ages of 5 AS patients at diagnosis were from 2 to 12 months. All AS patients (100%) showed seizures which were similar to that reported previously [19]. The apparent happy disposition, such as inappropriate laughter and excitability, was the other unique behavior of the AS patients and noticeable for pediatricians to suspect that child had AS and suggest further genetic tests. Of 5 AS patients, 3 older AS patients at the age of about 1 year old showed happy disposition, whereas other 2 patients aged from 2 to 3 months were too young to have such performance. For PWS, the most significant features are hypotonia in infancy followed by hyperphagia and life-threatening obesity [20], which are overlapped with other diseases during infancy, such as myotonic dystrophy, spinal muscular atrophy and so on [20][21][22][23]. As the result, early diagnosis is hard to make and 21 (80.77%) PWS patients were over 4 years old at diagnosis in the cohort of 26 PWS patients. Interestingly, one case (the patient 6) with mUPD was found in 31 PWS/AS (3.6%). She was taken to see a pediatrician because of severe intellectual disability and dysmorphic face. The G-banding karyotyping was initially performed for her showing a polymorphism located in 15ps (not shown) and was highly suspicious of PWS, so the further genetic tests were ordered. After the STR linkage analysis and then validated by MS-PCR and array CGH, she received a diagnosis of PWS with mUPD.
To our knowledge, this is the first time using a multiplex-fluorescent-labeled STRs assay to replace the sequencing-based STR analysis for identification of PWS/AS. Its major advantage over current techniques is the ability to identify near a variety of mutations with three multiplex.The innovative STR analysis provides an accurate, rapid and economic detection method for PWS/AS. It is also the optimal selection to identify the exact molecular defect about genetic imprinting disorders, which is valuable for genetic diagnosis, management of the pediatric patients and improvement of prognosis.