An Atypical 15q11.2 Microdeletion Not Involving SNORD116 Resulting in Prader–Willi Syndrome

Loss of expression of paternally imprinted genes in the 15q11.2-q13 chromosomal region leads to the neurodevelopmental disorder Prader–Willi Syndrome (PWS). The PWS critical region contains four paternally expressed protein-coding genes along with small nucleolar RNA (snoRNA) genes under the control of the SNURF-SNRPN promoter, including the SNORD116 snoRNA gene cluster that is implicated in the PWS disease etiology. A 5-7 Mb deletion, maternal uniparental disomy, or an imprinting defect of chromosome 15q affect multiple genes in the PWS critical region, causing PWS. However, the individual contributions of these genes to the PWS phenotype remain elusive. Reports of smaller, atypical deletions may refine the boundaries of the PWS critical region or suggest additional disease-causing mechanisms. We describe an adult female with a classic PWS phenotype due to a 78 kb microdeletion that includes only exons 2 and 3 of SNURF-SNRPN with apparently preserved expression of SNORD116.

While the importance of paternally expressed genes in the development of PWS pathology has been appreciated for many years, the identifcation of specifc critical genes associated with the phenotype has proved challenging.Small nucleolar RNA (snoRNA) genes that are under the control of the SNURF-SNRPN promoter, namely, the SNORD116 snoRNA gene cluster, have been implicated directly in disease etiology [3,6].Tis SNRPN gene is responsible for encoding the bicistronic SNURF-SNRPN transcript, hence why the genes are typically annotated together [2].Multiple reports of microdeletions including the SNORD116 snoRNA gene cluster have been found in patients with classic PWS in conjunction with normal methylation patterns in this region, thus implying that the SNORD116 gene cluster is a minimally critical PWS region [2,[7][8][9][10][11][12][13].Tere are some case reports that suggest that the PWS critical region is more complex than this isolated snoRNA gene cluster [14][15][16].Newer evidence points to a more nuanced understanding in which both SNORD116 and SNURF-SNRPN distinctly contribute to diferent components of the PWS phenotype [17,18].
Here, we report a de novo microdeletion within 15q11.2 in a woman with classic manifestations of PWS.Te deletion included only exons 2 and 3 of SNURF-SNRPN, and we demonstrate expression of SNURF-SNRPN cDNA.We also found preserved expression of several snoRNA clusters including SNORD116, the foremost postulated PWS critical region [13].No expression data was conducted on other genes within the region.Tis case indicates that the PWS phenotype is possible through a proximal loss of SNURF-SNRPN and challenges the paradigm that loss of active snoRNA gene clusters, specifcally SNORD116, is required for phenotypic manifestation of PWS.

Patient Report
A 23-year-old woman was evaluated for the frst time in a medical genetics clinic for an intellectual disability.Prenatally, she was noted to have fetal polyhydramnios, followed in the neonatal period by severe hypotonia and feeding difculties requiring syringe feeding.Tis continued in childhood as a persistent failure to thrive.By the age of 8 years, she developed hyperphagia, nonsatiety, and food hoarding behaviors that have continued into adulthood.She exhibited delayed puberty and mild developmental delays in all milestones with greater defcits in speech and gross motor skills.Her IQ was low-normal per clinical documentation, requiring an individualized education plan in school.During childhood, she showed signs of abnormal behaviors including sensory integration issues, skin picking, anger outbursts, and sleep difculties (waking three times per night).She also had highly viscous saliva and experienced difculty in articulating words, described as "mumbling."Te patient completed high school with supportive services and pursued vocational training as an adult after completing an associate degree.Family history was negative for any other individuals with signs of PWS.At clinical presentation (23 years old), her height was 150 cm (2 nd %ile), weight was 95.5 kg (99 th %ile), and body mass index (BMI) was 42.52 kg/m 2 .She was noted to have central obesity.Her skin was fair with light blonde hair.Her skin had red punctate scabs over her forearms from skin picking.Her facial features (Figure 1) included almondshaped eyes, mild left esotropia, and a small mouth with downturned corners.She did not resemble her parents.She had small hands and feet with normal fnger and toe morphology.A musculoskeletal exam revealed low tone throughout.She was otherwise neurologically typical aside from the observed skin picking, social anxiety and nervousness, and avoidance of eye contact.Te patient's overall PWS clinical score was 10 (maximum of 13.5 points,

Materials and Methods
3.1.Clinical Testing.Methylation analysis for the PWS/ Angelman Syndrome region was performed clinically by ARUP Laboratories (Salt Lake City, UT) using a standard methylation sensitive polymerase chain reaction/fuorescence monitoring assay (Roche Molecular Systems, Inc.).Per ARUP, this test has a sensitivity of over 99% for detecting PWS caused by methylation defects.Chromosomal microarray (CMA) was performed clinically by the Colorado Genetics Laboratory of the University of Colorado Denver on DNA extracted from patient's peripheral blood and hybridized with same-sex normal reference DNA using the CytoChip 180k Oligo Array platform (Illumina, Inc).Fluorescence in situ hybridization (FISH) analysis was performed clinically on samples from the patient and both parents by the Colorado Genetics Laboratory of the University of Colorado Denver, using BAC clone RP11-125E1, which hybridizes to the PWS region on 15q11.2.SNURF-SNRPN expression studies were performed at Stanford Genetics Laboratories with standard amplifcation of SNURF-SNRPN exons 9 and 10 using complementary DNA extracted from peripheral blood leukocytes [20].
Te patient and her parents gave permission for the additional studies based on our genetic research protocol (#07-0516) which was reviewed and approved by the Colorado Multi-Institutional Review Board.Tey also provided written consent for the publication of the clinical information and photographs.

2
Case Reports in Genetics

Case Reports in Genetics
Reverse transcription-PCR was performed using primers that spanned the coordinates chr15 : 25200167-25223718 (hg19).Te start coordinate of the frst primer was within intron 5 of transcript NM_001400738, and the end coordinate of the second primer was within the exon 14 of transcript NM_001400738.Primer sequences are available upon request.
In Figure 2, hg19 RefSeq-curated transcripts with exon coordinates on chromosome 15 were downloaded from the UCSC genome browser and loaded into R version 4.0.3.Visualizations of the data were generated using ggplot2 version 3.3.5.Exons overlapping with the coordinates spanning any of the deletions listed in the lower half of the fgure were then selected.RefSeq accession numbers were subsequently mapped to gene symbols using the gene table from NCBI datasets (https://www.ncbi.nlm.nih.gov/datasets/tables/genes/).To ensure that all gene locations were mapped in a standardized manner, the longest RefSeq transcript was selected and then visualized.Select transcripts of clinical importance were annotated in Figure 2. A list of the transcripts utilized in this visualization is available in Supplementary Table 1.

Results
Molecular studies in this patient diagnosed with classic PWS revealed a normal PWS methylation pattern and an atypical 15q11.2deletion by CMA analysis.Based on the CMA (Figure 3(a)), the deletion size was initially estimated at 132 kb and spanned linear positions 25,092,034−25,224,089 (NCBI human genome reference assembly Build 37 (hg19)).Te deletion was confrmed by FISH (Figure 3(b)) to be present in the patient (by a diminished signal consistent with a partially adherent FISH probe) and absent in both parents (fully adherent FISH probe).Sequencing and SNP mapping refned the deletion and narrowed the region to 78 kb between two heterozygous SNURF-SNRPN SNPs: a novel SNP in intron 2 and the rs61999138 SNP.Te novel SNP was detected in both parents.Gene expression analysis of SNURF-SNRPN in cDNA from the patient's peripheral blood lymphoblasts with a clinical assay revealed intact exons 9 and 10 (Figure 4(a)).In addition, RT-PCR molecular studies from the patient's saliva demonstrated preserved expression of SNURF-SNRPN exons 6 through 13, as well as PAR5 and SNORD116.SNORD116 expression in the patient was confrmed with targeted RT-PCR and gel electrophoresis (Figure 4(b)).Figure 2 demonstrates the novel location of the deletion detected in this patient with classic PWS, the regions implicated, and other regions of interest as outlined in the literature.

Discussion
Our case provides additional evidence of the complexity of the minimal critical deletion regions that result in PWS.Our patient presented with most of the major and minor characteristics observed clinically in PWS (Table 1).A previously undescribed microdeletion was identifed in the 15q11.2region [4,5].Molecular tests revealed a normal methylation pattern at the SNRPN locus, but CMA and SNP homozygosity analysis revealed a 78 kb deletion that included SNURF-SNRPN exons 2 and 3. FISH analysis targeting the 78 kb region confrmed a deletion in the patient; parental studies showed a normal FISH pattern and no deletion.Tis small deletion size (78 kb) contrasts with the larger type I and type II deletions classically associated with PWS that are 6.0 Mb and 5.6 Mb, respectively [10].Type I and II deletions can usually be detected by testing for the absence of expression of SNURF-SNRPN exons 9 and 10.In the patient described here, exons 9 and 10 of these genes in addition to the SNORD116 gene cluster were present and expressed.Importantly, a diagnosis of PWS may be missed if testing stopped with a negative test targeted for SNURF-SNURPN exons 9 and 10.If clinical presentation is consistent for PWS and a routine clinical test is negative, it is critical to pursue additional testing, such as CMA, to evaluate for less common genetic causes of PWS.
Ultimately, it has been challenging to determine the exact genes implicated in the pathogenesis of PWS.However, one leading theory is that SNORD116 is the minimal critical region for the PWS phenotype [13].Tis was ascertained by determining the smallest overlapping region of atypical deletions found in patients with classic PWS [2,[8][9][10][11].Deletions described in the literature that include SNORD116 and are associated with PWS only sometimes include SNURF-SNRPN.Of the eight deletions reported that include SNORD116, three also include all or part of SNURF-SNRPN [7,9,10].Tese three studies that included SNURF-SNRPN reported patients with a wide array of symptoms, from those with only a few features of PWS to those with classic PWS.In addition, the fve studies that report   Case Reports in Genetics deletions including SNORD116 but sparing SNURF-SNRPN also report a wide spectrum of phenotypes with these deletions [2,8,11,13,21].Tus, there does not seem to be a genotype-phenotype correlation among patients with deletions that spare SNURF-SNRPN.Despite this replicated evidence, other reports point to a more complex picture, especially when exons near the imprinting center are afected.For example, one of the smallest atypical deletions is described in a patient with many of the PWS characteristics [14].Tey describe a 6.4 kb deletion that overlaps with the imprinting center in a patient with decreased fetal movement, poor feeding, and hypotonia in infancy who developed developmental delays and obesity with small hands and feet (consistent with nonclassic PWS given that the clinical score was less than 8).In another case series (N � 8) of atypical deletions in patients with PWS, one of the patients also had a partial deletion of SNURF-SNRPN (including the entire SNURF transcript (NM_005678.5) and exons 4-13 of SNRPN (NM_001400738.1);see Figure 2) that did not directly afect SNORD116 [18].Tis patient had infantile hypotonia, obesity, hyperphagia, behavioral challenges (irritability), and skin-picking and did not require tube feeding as an infant; thus, this patient also did not meet enough criteria to be consistent with classic PWS.
Tere are two reports of patients with variants in SNURF-SNRPN who have features of PWS.Te frst is a patient with a missense variant in SNURF-SNRPN (c.193C > T, p.Arg65Trp) as well as a high degree of homozygosity that afected many genes in addition to part of SNURF-SNRPN [15].Tis patient had hypotonia and poor feeding in infancy with decreased fetal movement and developed hyperphagia, obesity, small hands, and endocrine abnormalities.Functional studies show the overexpression of SNURF-SNRPN, possibly resulting in a dominant negative efect, but they also demonstrate normal RNA expression of SNORD116 [15].Huang et al. also recently reported a mosaic, nonsense SNRPN variant (c.73C > T, p.R25X) found in a patient with some fndings of classic PWS, although with an overall milder form of the phenotype [17].Te present study is now the fourth published patient with classic PWS fndings that does not demonstrate an aberration of either an exon near the imprinting center of SNURF-SNRPN nor SNORD116 [15,17,18].Te deletion found in our patient is also unique without any overlapping deletion reported in the literature.

Conclusion
We demonstrate a unique molecular presentation in a patient with classic PWS associated with a 78 kb microdeletion involving exons 2-3 of the SNURF-SNRPN gene and preserved expression of downstream SNORD116.Tis result shows that classic PWS is not solely dependent on absent SNORD116 expression and that more work is needed to understand the mechanisms driving the phenotype.Tis study further demonstrates the importance of considering atypical microdeletions as a mechanism of PWS when initial methylation studies are normal.Our work also highlights that both methylation testing and CMA may be needed to ensure that a molecular diagnosis of PWS is not missed, particularly when clinical suspicion is high.

Figure 1 :
Figure 1: Clinical photographs of present case.Clockwise from top left: front photo of face demonstrating elevated BMI, facial features including narrow appearing face, almond-shaped eyes, thin upper lip, fair skin and hair, mild left esotropia.Feet photo demonstrating small feet.Hands photo demonstrating small hands.

Figure 2 :
Figure 2: Visualization of chromosomal microdeletions at 15q11.2-q13.3associated with PWS.Te top half of this fgure demonstrates a schematic of the 15q11.2-q13.3locus (not to scale).Te classic type I and II deletions (6 Mb and 5.6 Mb, respectively) associated with PWS are shown for reference as grey bars.Te PWS and Angelman syndrome (AS) regions are highlighted in yellow and blue, respectively.Breakpoints (BP) are also indicated.Te lower half of the fgure demonstrates a 420 kb region that contains microdeletions reported in the literature that are adjacent to the deletion described in this paper.Deletions associated with a classic PWS phenotype are shown in dark grey, and those associated with a nonclassic PWS phenotype are shown in light grey.Te red box denotes the location of the deletion described in this paper, which includes exons 2 and 3 of SNRPN (NM_001400738.1).Te gene symbols associated with select transcripts of clinical importance are displayed.Te term "SNURF-SNRPN" is utilized here for consistency with prior literature and refects the bicistronic nature of the transcript.

Figure 3 :
Figure 3: Fluorescence in situ hybridization (FISH) and chromosomal microarray.(a).Chromosomal microarray results of proband indicating ∼132 kb deletion at 15q11.2 (red line).(b).Analysis by FISH with cloneRP11-125E1 (red signal) confrmed partial deletion at 15q11.2.Blue probe is the centromere of chromosome 15, and the green probe is the control PML probe of 15q22.FISH, fuorescence in situ hybridization.

Table 1 )
, well above the clinical score criteria of 8 or greater established for diagnosis of PWS [4, 5, 19] based on the well-established defnition by Holm et al.Her clinical diagnosis of classic PWS is distinguished from a diagnosis of nonclassic PWS, in which a patient has some features of PWS but does not have a clinical score of at least 8.