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Am J Med Genet A. Author manuscript; available in PMC 2019 Aug 9.
Published in final edited form as:
Am J Med Genet A. 2018 Apr; 176(4): 886–895.
Published online 2018 Feb 13. doi:10.1002/ajmg.a.38627
PMCID: PMC6688622
NIHMSID: NIHMS1043081
PMID: 29437285
Samantha N. Hartin,1 Waheeda A. Hossain,1 Nicolette Weisensel,2 and Merlin G. Butler1
Author information Copyright and License information PMC Disclaimer
The publisher's final edited version of this article is available at Am J Med Genet A
Abstract
Prader–Willi syndrome (PWS) is a complex genetic imprintingdisorder characterized by childhood obesity, short stature,hypogonadism/hypogenitalism, hypotonia, cognitive impairment, and behavioralproblems. Usually PWS occurs sporadically due to the loss of paternallyexpressed genes on chromosome 15 with the majority of individuals having the15q11-q13 region deleted. Examples of familial PWS have been reported butrarely. To date 13 families have been reported with more than one child with PWSand without a 15q11-q13 deletion secondary to a chromosome 15 translocation,inversion, or uniparental maternal disomy 15. Ten of those 13 families wereshown to carry microdeletions in the PWS imprinting center. The microdeletionswere found to be of paternal origin in nine of the ten cases in which familystudies were carried out. Using a variety of techniques, the microdeletions wereidentified in regions within the complex SNRPN gene locusencompassing the PWS imprinting center. Here, we report the clinical and geneticfindings in three adult siblings with PWS caused by a microdeletion in thechromosome 15 imprinting center inherited from an unaffected father thatcontrols the activity of genes in the 15q11-q13 region and summarize the 13reported cases in the literature.
Keywords: chromosome 15, familial imprinting center, microdeletion, Prader-Willi syndrome (PWS)
1 |. INTRODUCTION
Prader–Willi syndrome (PWS) is a complex genomic imprinting disordercaused by a number of different genetic mechanisms leading to the loss of geneexpression from the paternal chromosome 15. The most common defect is a deletion ofthe chromosome 15q11-q13 region historically reported to occur in about 70% ofcases, followed by maternal uniparental disomy (both chromosome 15s inherited fromthe mother) in 30%. The remaining patients have an imprinting center defect eitherdue to a microdeletion or epimutation. Two typical PWS deletion subtypes (Type I orType II) are commonly reported, while atypical deletions which may be larger orsmaller in size than the typical deletions are less common. More clinicaldifferences are seen in those with the typical larger 15q11-q13 Type I deletioncompared with the smaller Type II deletion or maternal disomy 15 (; ; Zarcone et al., 2007). PWSwas the first reported human disorder found to be caused by errors in thedifferential expression of genes depending on the parent of origin or genomicimprinting.
PWS is characterized by failure to thrive and feeding difficulties in infancyand later, an uncontrollable appetite leading to obesity. Short stature,hypogonadism, hypotonia, and cognitive/behavior problems are common features. Theincidence is one in 10,000–30,000 with most cases due to sporadic events;however, 13 familial cases of PWS have been reported (; ; Buiting et al., 2000; ; ; ; Jancar, 1971; Lubinsky etal., 1987; ; Ming et al., 2000;Ohta et al., 1999; Orstavik et al., 1992; Reis et al., 1994; Teshima et al.,1996). Historically, the first familial cases were reported in 1971 intwo affected brothers with PWS, one having a dizygotic twin sister without PWS(Jancar, 1971). They had normalchromosome studies at that time prior to the discovery of the chromosome 15q11-q13deletion reported in 1981 (Ledbetter et al.,1981). The mother was reported to have a below average intelligence buttheir father and two other siblings were reported as physically and cognitivelynormal.
In the 13 families reported, the subjects met the major and minor criteriafor a clinical diagnosis of PWSas proposed by Holmet al. (1993). The major criteria are neonatal hypotonia, feedingproblems, rapid weight gain, characteristic facial features (narrow face,down-turned mouth), hypogonadism, developmentaldelay, hyperphagia, and cytogeneticabnormalitiesin the 15q11-q13 region. The minor criteria are decreased fetalmovement, characteristic behavioral problems (tantrums, obsessive-compulsivebehavior), sleep disturbances, short stature, hypopigmentation, small hands and/ orfeet, eye abnormalities, thick saliva, speech problems, and skin picking. Ten of the13 familial cases with PWS in the literature were found to have microdeletionsaffecting the imprinting center (; Buiting et al.,1995; Buiting et al., 2000; Ishikawa et al., 1987, 1996; ; Ming et al.,2000; Ohta et al., 1999; Orstavik et al., 1992; Reis et al., 1994; Teshima et al., 1996). The microdeletions were passed to the subjectsfrom the paternal grandmother through their father. Prader-Willi syndrome arises inthe grandchild because the microdeletion is passed directly to the offspring by thefather. The other three PWS families showed no chromosome 15q deletion (Burke et al., 1987; ; Jancar, 1971). However, microdeletions in those subjectscannot be ruled out as analysis of the PWS imprinting region was not carried out.Here, we report three adult siblings with PWS caused by a microdeletion in the PWSimprinting center within the 15q11-q13 region inherited from the father andsummarize relevant literature.
2 |. CLINICAL REPORTS
2.1 |. Subjects
2.1.1 |. Sibling ABCF31
Sibling ABCF31 is a 40-year-old female with a history of PWSconfirmed at the age of 23 years via fluorescence in situ hybridization(FISH) using a 15q11-q13 SNRPN probe. She was born 4 weeks premature tonon-consanguineous parents. There were no complications during the pregnancybut decreased intrauterine movements were noted. She weighed 2.36 kg (2ndpercentile) with APGAR scores of 5 and 6. She was hypotonic, essentiallynever cried, and had a poor suck but did not require tube feedings. Sheremained hospitalized for 10 days after delivery secondary to elevatedbilirubin levels and feeding issues. She walked at 12 months and her verbalskills were considered normal. She had mild intellectual development delaywith full-scale IQ testing of 59 at the age of 17 years. Her appetite beganto increase at 4 years of age and was noted to be overweight by 7 years. Hermaximum weight was 138.8 kg (99th percentile) at the age of 26 years. She iscurrently 154.94 cm (10th percentile) in height and weighs 72.57 kg (82ndpercentile) while in residential care. She has a history of significantfood-seeking behaviors and has stolen food at home and from stores in thecommunity in the past.
She was diagnosed in the past with bipolar affective disorder type I,impulse-control disorder, depressive disorder, not otherwise specified, mooddisorder, not otherwise specified, personality change secondary to PWS, andnonverbal learning disorder. Her history of mood symptoms noted at 19 yearsof age included argumentativeness with manipulative behaviors. She hadbaseline demeanor changes with agitation, restlessness, irritable mood, andincreased rate of speech and was admitted to the Prader–Willi Homesof Oconomowoc (PWHO) in Wisconsin at 26 years of age. She has a history ofanxiety, skin picking, asking repetitive questions, verbal and physicalaggression, agitation, and rapid mood shifts. She made multiple suicidalthreats in the past, as well as threats to elope during adulthood. She alsodrank a bottle of body lotion during this one year period but did notrequire hospitalization following the ingestion. She was appropriatelymonitored and treated at PWHO by clinical staff under the care of apsychiatrist. Her family noted social withdrawal, sadness, and increasedsleep but she was not hospitalized for psychiatric problems. Her current DSMV psychiatric diagnoses include personality change secondary to PWS, bipolaraffective disorder type I, skin picking disorder, intellectual developmentaldisorder-mild, and learning disorder.
Additionally, she was diagnosed with morbid obesity, hypertension,and diabetes mellitus type II; all of which resolved with weight loss andexercise. She has also suffered from chronic sinusitis, chronicconstipation, acid reflux, enuresis, osteopenia, borderlinethrombocytopenia, and irregular menstrual spotting while on oralcontraceptives. Her medical history includes a nondisplaced oblique fractureof the base of a toe, levoscoliosis of the lumbar spine-mild to moderateleftward curve, moderate S-shaped thoracolumbar spine, leftward curvature ofmid-upper lumbar spine with subtle rotary component, a lumbar L1 compressionfracture, and degenerative disk disease.
2.2 |. Sibling ABCM26
Sibling ABCM26 is the 35-year-old brother of Sibling ABCF31 and ABCM51.He was diagnosed with PWS at 18 years of age. His mother received fertilitydrugs prior to conception. He was born 7 weeks premature with a double footlingbreech presentation but delivered vagin*lly. No complications were reportedduring the pregnancy. He weighed 2.18 kg (1st percentile) at birth, washypotonic and cried little, with a poor suck and feeding problems. He requiredorogastric tube feeds for the first 8 weeks of life and remained in the neonatalintensive care unit for 6 weeks after birth. A systolic heart murmur wasidentified shortly after birth which resolved by 2 years of age. He hadseizure-like activity on one occasion shortly after birth with a negativeneurologic workup and without reoccurrence.
He rolled over independently at 5 months and walked by 12 months. He wasable to produce four word sentences by 16 months; toilet trained by 3 years andbegan reading at a normal age. He had mild intellectual developmental delay withfull-scale IQ testing of 65 at the age of 18 years. Significant hypotoniacontinued and increased appetite began at 3 years of age. His weight gainincreased by 5 years of age and was overweight by 7 years. His maximum weight of120.2 kg (>99th percentile or 2.7 Z-score) occurred at 18 years of age.His current weight is 82.37 kg (80th percentile) with a current height of 157 cm(< 1st percentile or −2.7 Z-score).
He was diagnosed with major depressive disorder, depressive disorder,not otherwise specified, anxiety disorder, not otherwise specified,posttraumatic stress disorder, obsessive-compulsive disorder, oppositionaldefiant disorder, intermittent explosive disorder, personality change secondaryto a PWS, nonverbal learning disorder and a provisional diagnosis of mixedreceptive, and expressive learning disorder. His parents became concerned aboutdepressive symptoms including social withdrawal and sadness between the ages of11 and 12 years. He has an extensive history of self-harm which began at 13years of age including inserting objects under his skin, gouging, burning, andbiting himself at times to the point of tissue loss. A suicide attempt occurredat 17 years of age when he overdosed on acetaminophen and Motrin. He laterattempted suicide by hanging.
He has had multiple previous psychiatric hospitalizations with fouradmissions to The Children’s Institute (TCI) in Pittsburgh, PA. His firstpsychiatric hospitalization was at 16 years of age followed by three additionalpsychiatric hospitalizations at 17 years. At the age of 18, he was admitted toTCI secondary to excessive food-seeking behavior, morbid obesity, and ongoingdepressive symptoms with suicidal ideation. A second admission occurred at 19years of age for 45 days secondary to significant depressive symptoms includinga self-inflicted laceration. At discharge, he was referred toPrader–Willi Homes of Oconomowoc (PWHO) for residential care. He had twobrief hospitalizations at Winnebago Mental Health Institute in Wisconsin (WMHI)at the age of 19 years for depressive symptoms and suicidal ideation but thenhis mood appeared to stabilize without major incidents of stealing or self-harmuntil the age of 20 years. He was initially admitted to Rogers Memorial Hospitalafter jabbing a pen into his arm that required medical attention as well asdepressive symptoms and suicidal ideation. While at Rogers Memorial, he stabbedhimself with a fork and was subsequently transferred back to WMHI. He remainedthere for 2 months. He had another admission to WMHI at 21 years of age but wastransferred to TCI where he was hospitalized for 2 months for the third timesecondary to significant depression, suicidal ideation, and self-mutilation. Hewas admitted again at 21 years of age to TCI for significant depressivesymptoms, suicidal ideation, and self-mutilation. He also has a history ofpulling head and eyebrow hair in secret or when angry.
He has rare verbal outbursts, but displays intermittent skin picking andrectal digging. He has a history of significant food-seeking behaviors,consuming inedible objects (cigarette butts) and has stolen edible items fromstores, peers, and also his family. He broke into a freezer at hisparents’ home with his brother and stole food on at least one occasion.He also has a history of chronic kidney disease stage III with an atrophic leftkidney, degenerative disc disease, chronic constipation with a history of rectaldigging, enuresis, scoliosis, status post abdominal, and inguinal herniarepairs, osteoporosis, anemia, gastroesophageal reflux disease, positive PPD(purified protein derivative) but further workup was negative for tuberculosisand he had two subarachnoid hemorrhages at 25 years of age, secondary to a falland striking head on concrete. He required 6 weeks of inpatient rehabilitationand displayed a possible change in articulation since the injury, per parents.His current DSM V psychiatric diagnoses include personality change secondary toPWS, other specified depressive disorder, other specified obsessive-compulsive,and related disorder (repeatedly placing objects under skin and rectal picking),borderline intellectual function, learning, and language disorder and othermedication-induced movement disorder (neuroleptic-induced Parkinsonism andpotential lithium contribution to bilateral hand tremor).
2.3 |. Sibling ABCM51
Sibling ABCM51 is the33-year-old brother of Sibling ABCF31 and SiblingABCM26. His parents were 33 years old at conception. His mother was placed onbed-rest for the last trimester, secondary to history of previous prematurebirth. He was born 3 weeks premature and weighed 2.41 kg (3rd percentile) withAPGAR scores of 7 and 9. At birth he was hypotonic, but had a suck reflex anddid not require tube feedings or other feeding assistance. He remainedhospitalized for 6 days after birth secondary to elevated bilirubin levels. Hewas first diagnosed with PWS at the age of 16 years along with his two oldersiblings. He had a maximum weight of 161.4 kg (>99th percentile or 3.5Z-score) at 18 years and currently weighs 88.9 kg (89th percentile, and standsat 167.64 cm (11th percentile). He has a history of significant food-seekingbehaviors and has stolen edible items from his family at home.
He rolled over independently by 3 months, crawled at 8 months, andwalked at 11 months. He has the most delayed verbal/language skills of the threesiblings with difficulties in word retrieval. He was toilet trained by 2.75years of age. He had early intervention services: PT, OT, and speech therapy,beginning at approximately 4 years old until kindergarten. He had mildintellectual developmental delay with full-scale IQ testing of 70 at the age of16 years. His appetite began to increase at 5 years of age and at 9 years wasnoted to be overweight. His parents stated that he was more physically activeand stronger than his two older siblings and was more active in utero andcontinuing through childhood. He was more adventurous about exploring hissurroundings as a toddler, more likely to try new things and to take morephysical risks than his siblings. He is more physically active than the typicalindividual with PWS as he participated in downhill skiing and wrestling inmiddle school.
His parents became concerned at approximately 12 years of age, due toanger, oppositional defiant disorder symptoms, and anxiety. His psychiatricsymptoms were not as severe as his two older siblings; however, he was admittedto The Children’s Institute (TCI) in Pittsburgh, PA at 18 years of age.His maximum weight occurred prior to this hospitalization. At discharge from TCIhe was then admitted to PWHO, which was his first group home admission.
He has an extensive history of verbal aggression, threatening group homestaff (particularly female staff) and family members. He has significanttantrums, rare impulsive suicidal ideation, minimal skin picking, hoarding, andelopement. He has not been hospitalized solely for psychiatric reasons nor hashe had a history of suicide attempts or psychotic symptoms. He was previouslydiagnosed with attention deficit hyperactivity disorder, oppositionaldefiant—disorder, anxiety disorder secondary to a general medicalcondition, personality change secondary to PWS, nonverbal learning disorder− mixed, expressive, and receptive language disorder, borderlineintellectual functioning and mild intellectual developmental delay. His currentDSM V psychiatric diagnoses include personality change secondary to PWS,generalized anxiety disorder, oppositional defiant disorder, attention deficithyperactivity disorder, borderline intellectual function, and learning andlanguage disorder.
At 3 years of age, he underwent surgery for bilateral undescended testesand an inguinal hernia. His medical history also includes an overactive bladderand urinary incontinence, idiopathic thrombocytopenic purpura, lower back pain,patellofemoral chondrosis with resultant chronic left knee pain, chronicconstipation, gastroesophageal reflux disease (GERD), and sleep apnea. He wasprescribed CPAP for sleep apnea but refused treatment. He also has diabetesmellitus type II and chronic nasal congestion.
3 |. GENETIC TESTING METHODS
3.1 |. DNA collection and extraction
All individuals agreed to participate and signed informed consent formsapproved by the institutional review board at the University of Kansas MedicalCenter before entry into the study. Saliva was collected using a Saliva DNACollection and Preservation Device (Norgen Biotek Corporation, Thorold, Ontario,CA) and genomic DNA isolated and purified using a Saliva DNA Isolation Kit(Norgen Biotek Corporation) according to manufacturer’s protocol.
3.2 |. Methylation specific-multiplex ligation probe amplification(MS-MLPA®)
MS-MLPA reagents and kits were obtained from MRC-Holland (Amsterdam, theNetherlands) and MS-MLPA carried out on all three siblings and their parentsusing the ME028-B2 kit containing sequence-specific probes along the length ofthe 15q11.2-q13 region. The B2 kit contains 48 MLPA probes for copy numberdetection and methylation status using methylation-sensitive restrictionenzymes. Approximately, 50 ng of genomic DNA was used for each of the MS-MLPAreactions and carried out according to manufacturer’s instructions. TheMS-MLPA PCR products were sent to GENEWIZ (Frederick, MD) for fragment analysis,a commercially available sequencing laboratory for a fee for service.
3.3 |. High-resolution microarrays
High-resolution microarray analysis was carried out on subjects ABCM51and ABCF31 using an Affymetrix™ Genome-Wide Human SNP Array 6.0(Affymetrix, Inc., Santa Clara, CA) performed on 3 μg of intact genomicDNA undertaken at the Genomics Core facility at KUMC. Genomic DNA from ABCM51was also analyzed using an Infinium Omni5Exome-4 BeadChip array (Illumina, Inc.,San Diego, CA) by Victorian Clinical Genetics Services at the MurdochChildren’s Research Institute in Victoria, Australia.
3.4 |. Digital droplet PCR (ddPCR)
Digital droplet PCR (ddPCR) studies were performed on the subjects andtheir parents in triplicate to confirm the location of the imprinting centerdeletion. Three different primers were designed and FAM-tagged probe sets werelocated in the PWS imprinting center on chromosome 15 (IC1, IC2, and SRNPN exon1). The primers and FAM tagged probe sets were purchased from Integrated DNATechnologies (Coralville, IA). We also purchased validated GABRβ3 HEXddPCR copy number assay probe from Bio-Rad (UniqueAssayIDdHsaCP2506453-Hercules, CA)touse as a reference, as it is outside the PWSimprinting center but in the 15q11-q13region. Prepared reactions were undertakeninthe Genomics Core at the University of Kansas–Lawrence. Data wereanalyzed using the QuantaSoft Analysis Pro software available from Bio-Rad.
4 |. RESULTS AND DISCUSSION
Thirteen families were previously reported in the literature with more thanone child with PWS and without a recognized 15q11-q13 deletion secondary to achromosome 15 translocation, inversion, or uniparental maternal disomy 15 (Table 1). Three of the families were diagnosedwith PWS before the use of high-resolution chromosome analysis. However, when datawere available in the literature reports, the subjects met the major and minorconsensus diagnostic criteria for PWS (Table2). Jancar (1971) reported on twobrothers with PWS; both were hypotonic at birth, had developmental delays,hypogonadism, and acromicria, were of short stature and had obesity at a young age.The older brother was reported to have feeding difficulties, high myopia, and dryoral mucosa and to be restless at times. His diagnostic score was 7.5 and youngerbrother’s score was 5.
TABLE 1
Diagnostic tests and results from studies of 13 cases of familialPrader-Willi syndrome
| Reference | Subjects | Testing/Results |
|---|---|---|
| Jancar(1971) | Two PWS brothers clinically diagnosed. One hadan unaffected female dizygotic twin. | Chromosomal investigation displayed normalmale karyotypes. Done prior to high-resolution chromosome analysis. |
| 1. Hall andSmith (1972) 2. Clarren and Smith (1977) | Two PWS male maternal first cousins clinicallydiagnosed. | Diagnosed clinically only. Done prior tohigh-resolution chromosome analysis. |
| Burke et al.(1987) | Three PWS sisters clinically diagnosed andfour unaffected siblings. | All three affected sisters, an unaffectedsister and their mother, had normal karyotypes by high-resolutionchromosome analysis. |
| 1. Lubinsky etal. (1987) 2. Buitinget al. (1995) | 1. Four PWS siblings clinically diagnosed (twofemales, one died at 10 months from pneumonia, and two males). 2. Twoaffected brothers (family “U“ in Buiting et al., 1995). | 1. High-resolution chromosome analysis showednormal karyotypes for all siblings and parents. 2. Quantitative DNAdosage analysis for 5’ SNRPN exons showed exons −1 and 0deleted but exon 1 was intact in both male siblings and father. |
| 1. Ishikawa etal. (1987) 2. Ishikawaet al. (1996) | 1. Two PWS sisters and one unaffected brother.2. Two siblings, no sex given (family “PWS-J“ in Ohta et al., 1999). | 1. One sister with a normal karyotype and onewith a terminal deletion of distal Xq. 2. SNRPN cosmid showed exons4–10 deleted. Fathers’ DNA not available. |
| 1. Orstavik etal. (1992) 2. Sutcliffe et al. (1994) | Family “O“. PWS brother andsister with an older brother who died at 7 days of age from respiratorydistress. | FISH analysis with SNRPN cosmid showed SNRPNdeletion in PWS siblings, father and paternal grandmother. |
| Reis et al.(1994) | Family “S“. PWS brother andsister. | Densitometry studies showed proximal SNRPNdeletion in both siblings and their paternal grandmother. |
| Teshima et al.(1996) | Two PWS brothers (“PWS-T“ inOhta et al., 1999). | FISH analysis with SNRPN cosmid showed SNRPNdeleted in siblings and father. |
| Ohta et al.(1999) | Family “PWS-P“. PWS male andfemale siblings. | Both had normal karyotypes. FISH analysis withSNRPN probe showed SNRPN deleted in siblings andfather. |
| Ming et al.(2000) | PWS male and female paternal firstcousins. | FISH analysis with SNRPN cosmid showed SNRPNdeleted in the cousins, their fathers and two paternal aunts. |
| McEntagart etal. (2000) | PWS brother and sister. | Microsatellite analysis showed SNRPN deletionin the siblings, their father and paternal grandmother. |
| Buiting et al.(2000) | Two PWS sisters. | Southern blot analysis identified deletion inthe imprinting center in the siblings, their father, an aunt, an uncleand their paternal grandmother. |
| Bingeliene etal. (2015) | Two PWS brothers and their PWS half-sister whodied of obstructive sleep apnea. | All three carried a 15q11-q13 paternalchromosome deletion. Reported paternal grandfather had a history of PWS(unconfirmed). |
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The deletions were paternal in origin in which genetic studies werecompleted in the above cases. PWS = Prader-Willi syndrome. FISH =fluorescence in situ hybridization.
TABLE 2
Consensus diagnostic criteria for Prader–Willi syndrome inindividuals with imprinting defects
| Three Siblings withPrader-Willi Syndrome | Bingeliene et al. [2015] | Buiting et al. [2000] | McEntagart et al. [2000] | Ming et al. [2000] | Ohta et al.[1999] | Teshima et al. [1996] | Reis et al. [1994] | Orstavik et al. [1992];Sutcliffe et al. [1994] | Ishikawa et al. [1987] and[1996] | Lubinsky et al. [1987];Buiting et al. [1995] | Burke et al. [1987] | Hall & Smith [1972];Clarren and Smith [1977] | Jancar [1971] | |||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ABC F31 | ABC M26 | ABC M51 | M 20yr | M 23yr | F* 24yr | F 5yr | F 2yr | M 10yr | F 6yr | M 0.5yr | F 16yr | F 4yr | M 3yr | M 7yr | M 12yr | M 29yr | F 26yr | M* 0.1 yr | M 12yr | F 7yr | F 22yr | F 19yr | F* 0.8yr | M 26yr | F 27yr | M 21 yr | F 26yr | F 22yr | F 26yr | M 17yr | M 22yr | M 22yr | M 20yr | |
| MajorCriteria | ||||||||||||||||||||||||||||||||||
| Neonatal hypotonia | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||
| Feeding problems | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||||||||||
| Excessive or rapid weight gain | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||||||||||||||||||||
| Characteristic face (narrow face, almondshaped eyes, down turned mouth) | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||||||||
| Hypogonadism | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||||||||
| Developmental delay | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | |||||
| Hyperphagia | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | |||||||||||||||||
| Chromosome 15q 11-q13 abnormality | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||||||||
| MinorCriteria | ||||||||||||||||||||||||||||||||||
| Decreased fetal movement | + | + | + | + | + | + | + | |||||||||||||||||||||||||||
| Characteristic behavior (tantrums, violentoutbursts, stealing) | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | |||||||||||||||||||
| Sleep disturbances | + | + | + | + | + | + | ||||||||||||||||||||||||||||
| Short stature | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | ||||||||||||
| Hypopigmentation compared to family | ||||||||||||||||||||||||||||||||||
| Small hands/feet | + | + | + | + | + | + | + | + | + | + | + | + | + | |||||||||||||||||||||
| Eye findings (esotropia, myopia) | + | + | + | + | + | + | ||||||||||||||||||||||||||||
| Thick saliva | + | + | + | + | + | + | + | |||||||||||||||||||||||||||
| Speech problems | + | + | + | |||||||||||||||||||||||||||||||
| Skin picking | + | + | + | + | + | + | + | + | + | |||||||||||||||||||||||||
| Total** | 10 | 10 | 9.5 | 10 | 10 | 2 | 7.5 | 6.5 | 9 | 8.5 | 7 | 7.5 | 6 | 7 | 4 | 7 | 5.5 | 5.5 | 1.5 | 10 | 9 | 7.5 | 7 | 3 | 9.5 | 9 | 9 | 8 | 7.5 | 7.5 | 7 | 6 | 7.5 | 5 |
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*Died prior to data analysis or incomplete data. M= male; F =female
**Diagnostic scoring criteria from Holm et al. (1993). Major criteria are worth one point each.Minor criteria are worth half point. A total score of five points arerequired for diagnosis of children with PWS at 3 years of age or younger.Four of which should come from the major group. A total score of eight isnecessary for diagnosis of PWS at 3 years of age to adulthood. Five or morepoints should come from the major group. Other features of PWS noted but notdiscussed.
Two male maternal first cousins were reported by Hall and Smith in 1972 andagain by Clarren and Smith in 1977 with reduced fetal movement, hypotonia at birth,developmental delays, hyperphagia, hypogonadism, small feet, and characteristicfacial features. The diagnostic score was 6 in the older cousin and 7 in the youngercousin. The three sisters reported by Burke et al. in 1987 were hypotonic at birth,gained weight rapidly, had hypogonadism, developmental delay, short stature, anddysmorphic facial features characteristic of PWS. Their diagnostic scores were 7.5,7.5, and 8.
Ten of those 13 families carried microdeletions in the PWS imprinting center(Table 1). Family studies were carriedout in 9 of the 10 cases and the microdeletions were found to be of paternal origin.These microdeletions included regions within the complex SNRPN genelocus encompassing the PWS imprinting center and identified by using a variety oftechniques. The microdeletions resulted in altered maternal and paternal imprintsduring gametogenesis as the paternally expressed genes could not be activated due tothis imprinting center microdeletion (Buiting et al.,1995). In seven of the families, the father carried the same SNRPNdeletion on his maternal chromosome 15 and in four families; the paternalgrandmother carried the SNRPN deletion on her maternally contributed chromosome 15.These data further support that lesions in the PWS imprinting center can change theepigenotype on chromosome 15 from a paternal epigenotype to a maternal epigenotypein regards to methylation and gene expression. Failure to erase the imprint makes itimpossible to set a new correct imprint in the germline. The maternal imprint couldnot be erased in the paternal germline in those with PWS imprinting centerdeletions.
PWS caused by an imprinting center defect falls within two classes:microdeletion or epimutation. Patients with PWS and epimutations on the paternalchromosome will not switch the imprint throughout the imprinted area in the15q11-q13 region. This failure to switch the imprint is a prefertilizationimprint-switch error since the imprinting center has no known function in somaticcells; however, a postzygotic event cannot be excluded (Buiting et al., 1998, 2003; Bürger et al., 1997;El-Maarri et al., 2001; ; ). An analysis of 51 PWSsubjects with an imprinting defect found that 86% of the subjects carriedepimutations and not microdeletions occurring spontaneously without other DNAchanges. The absence of other DNA mutations may indicate that the PWS imprintingcenter contains several redundant elements or can withstand sequence changes.Epimutations show biparental inheritance of chromosome 15 DNA markers, PWSDNA-methylation, and gene-expression patterns throughout the imprinted domain withinthe 15q11-q13 region (Buiting et al., 1995;Glenn et al., 1993; Reis et al., 1994; Saitohet al., 1996; Sutcliffe et al.,1994). Although epimutations have been reported to account for thelargest percentage of imprinting defects, the recurrence risk appears to benegligible (Buiting et al., 1998, 2003). However, when PWS results from amicrodeletion in the imprinting center carried by the father, the recurrence risk is50% (Buiting et al., 2000, 1995). If the deletion is only passed through thematernal line, then no phenotypic effect will occur but her son(s) would be at a 50%risk of having children with PWS. Her daughters’ sons would also be at riskof having children with PWS. Here, we provide an example of PWS in three siblingswho inherited a microdeletion in the PWS imprinting center on chromosome 15 fromtheir father. Previous DNA testing of the paternal grandmother showed a defect ordeletion on chromosome 15 that was found in the father in the imprinting center andpassed to the siblings with PWS. The father has two siblings; one sister withoutchildren and one brother with two daughters. The father also had three maternalaunts, one paternal aunt and two paternal uncles. There were no other individualsaffected with PWS.
Our PWS siblings demonstrate the dysmorphism and clinical features withbehavioral problems seen in PWS (Table 2). Atbirth all three siblings were hypotonic, one of the major criteria for a PWSdiagnosis. Siblings ABCF31 and ABCM26 had a poor suck but did not require tubefeedings. The youngest sibling, ABCM51, had a suck at birth but no other feedingproblems reported. The siblings were developmentally delayed and had onset ofchildhood obesity, short stature, hypogonadism, cognitive dysfunction andcharacteristic behavioral problems including skin picking, depression, food-seeking,and stealing with obsessive-compulsive behaviors. All three siblings exhibitedcharacteristic dysmorphic facial features-narrow head, a small down-turned mouth,almond-shaped eyes and strabismus, and central obesity (Figure 1).
FIGURE 1
Clinical photographs of the three siblings with PWS. a) ABCF31, 40 yearold female. b) ABCM26, 35 year old male. c) ABCM51, 33 year old male. Thesiblings display the typical characteristic facial features of PWS including anarrow face, almond-shaped eyes and a small down-turned mouth and centralabdominal obesity.
The PWS imprinted genes are only methylated on the maternally contributedallele and not on the paternal allele; therefore, the fragments from the genes inthis region are digested by about 50% in unaffected individuals during MS-MLPAanalysis. The MS-MLPA B2 kit contains five probes that are about 50% digested incontrol samples, including four from the SNRPN region. The DNA fragments fromimprinted genes on chromosome 15 are 100% methylated in PWS subjects regardless ofthe genetic subtype (Henkhaus et al., 2012).MS-MLPA of the siblings demonstrated the typical 100% methylation pattern for PWS asshown in Figure 2. The father displayed 0%methylation for the four probes in the SNRPNregion(Figure 2). Since the normal methylation signal from the active paternalallele is 0% and 100% from the maternal allele, the unaffected father should displaya normal 50% methylation pattern. However, he displayed 0% methylation for theprobes in the SNRPN region, indicating a SNRPNdeletion including the imprinting center on his maternal allele and 0% methylationpattern for his active paternal allele (). The MS-MLPA copy number analysis of all three siblings andtheir father detected a heterozygous deletion of the SNRPN regionin the PWS imprinting center (Figure 3). Twelveprobes from the MS-MLPA B2 kit detected a partial deletion of the SNRPN locuscontaining the imprinting center and ranged from SNRPN intron u2through the SNRPN locus and SNORD 116 snoRNAcluster located at 15q11.2. This analysis excluded a large deletion or uniparentaldisomy 15 and classified the PWS siblings as having an imprinting defect due to amicrodeletion. A normal methylation pattern and copy number were observed in theirmother (Figure 3).
FIGURE 2
Methylation patterns generated using Methylation Specific-MultiplexLigation Probe Amplification B2 kit (MS-MLPA-B2). The mother displayed a normalmethylation pattern for the four methylation sensitive fragments digested in theSNRPN region (in bold). The father displayed an abnormal methylation pattern(0%) in the SNRPN region due to loss on his maternal chromosome 15. All threesiblings (only ABCF31 shown) displayed the typical PWS methylation pattern(~80–100%) of the four SNRPN probes. UBE3A exon 1 and one otherdigestion control probe were used during methylation analysis.
FIGURE 3
Copy number generated using MS-MLPA-B2 kit. The mother has a normal copynumber of 2 for all gene fragments analyzed of chromosome 15. The father and allthree siblings (only ABCF31 shown) were deleted (showing a copy number of 1) for12 probes in and around the SNRPN region (triangles).
High-resolution microarray analysis was also performed on genomic DNA in twoof the siblings using the Affymetrix 6.0 SNP array. We identified microdeletions inthe imprinting center for ABCM51 and ABCF31. ABCF31 had a microdeletion located atthe 15q11.2 region (145 kbp) that includes SNURF-SNRPN andSNORD116 while ABCM51 carried a somewhat smaller deletion(129kbp). The Illumina 5 M microarray technology was also utilized and a 375 kbpmicrodeletion was found at 15q11.2 for ABCM51. The variation in size may be due tothe difference in the amount of probe coverage for each microarray platform. Neithermicroarray analysis was undertaken with sibling ABCM26 or the parents due to thecost of microarray testing.
Deletion status was also assessed by using digital droplet PCR (ddPCR) forvalidation of aforementioned results performed at the University of Kansas GenomicsCore facility with probes designed to detect microdeletions in the PWS imprintingcenter spanning chr15: 25,170,003–25,200,184 (hg19). All three PWS siblingsplus their father showed a copy number reduction using three probes with ddPCR,indicating that they carried a deletion in the imprinting region. The probes thatcovered an area within the imprinting center were: IC2 (hg19 chr15:25,170,055), IC1(hg19 chr15:25,181,550), and SNRPN exon1 (hg19 chr15:25,200,126). The PWSsiblings’ mother displayed a normal copy number for the same probes,indicating she did not carry a microdeletion in the PWS imprinting center (data notshown). Comparison of MS-MLPA and microarray analysis of DNA from the PWS siblingsverified the authenticity of our ddPCR results.
We encourage the reporting of other families with more than one affectedindividual with PWS not due to chromosome 15 trans-locations or inversions anddetailed genetic analysis to identify the genetic defect within the imprintingcenter and impact on phenotype. These studies will allow a better understanding ofthe cause and diagnosis of this rare obesity-related genetic syndrome.
ACKNOWLEDGMENTS
We thank the siblings and their parents for participation in this study. Wewould also like to thank Mandilea Bandt for collecting the saliva samples from thePWS siblings and Dr. Ann Manzardo for her comments on the manuscript. We acknowledgeNational Institute of Child Health and Human Development (NICHD-HD02528) andPrayer-Will Support PWS Organization (Family & Friends of KyleighEllington).
Funding information
National Institute of Child Health and Human
Development, Grant number: HD02528;
Prayer–Will Support PWS Organization
(Family & Friends of Kyleigh Ellington)
Footnotes
CONFLICTS OF INTEREST
There are no conflicts of interest to report for any of the authors.
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