Prader-Willi syndrome (PWS, MIM 17620) and Angelman syndrome (AS, MIM 105830) are genetic diseases caused by an imprinting defect of chromosome 15q11-13 [1, 2, 3, 4]. The estimated prevalence of PWS is 1 in 10,000 to 25,000 live births [3] and of AS is between 1 in 10,000 to 12,000 live births [2, 5]. Severe hypotonia and feeding difficulties in early infancy followed by weight gain and excessive eating in later childhood characterize PWS. Developmental delay, cognitive impairment (mean IQ of 60), hypogonadism, characteristic facial features, strabismus, scoliosis, short stature, behaviour and psychiatric problems are associated with PWS. Morbid obesity and type 2 diabetes mellitus are sequelae of the polyphagia, unless strict dietary controls are started in early life [6]. Growth hormone therapy is recommended for selected PWS cases [7]. Angelman syndrome is characterized by severe developmental delay, limited speech, gait ataxia and tremulousness of the limbs associated with a happy demeanour that includes inappropriate laughter. Seizures are common and associated with a high voltage activity on electroencephalography (EEG) [8]. In most cases, expressive speech is limited, and alternative, augmented communication is recommended and should be introduced early in affected cases [5]
The chromosome 15q11-13 is an imprinted region where only one parent’s copy of the gene is expressed with the second parental copy becoming methylated to transcriptionally inactivate it. The imprinting process, regulated by an imprinting centre (The PWS-IC is a 4.1-kb region, which spans the SNURF/SNRPN promoter and exon 1 and the AS-IC is an 880-bp sequence located ~ 35 kb centromeric of the PWS-IC) occurs in early post zygotic life and is an epigenetic mechanism for genetic disease. [9]
Around 65 – 75% of PWS [1, 10] and 60-75% of AS patients [2] have deletions involving chromosome 15q11-13. The common deletions are approximately 6Mb (type 1: 40% cases) and 5.3Mb (type 2: 60% cases) [10, 11, 12]. In PWS, the deletion involves loss of paternally expressed genes while the hemizygous 15q region contains the methylated (transcriptionally inactive) maternal genes. In AS, the maternal 15q contains the deletion with the hemizygous 15q region containing methylated paternal genes. Around 20 – 30 % of PWS cases [1] and 2-5% of AS cases [2] have uniparental disomy (UPD) as the mechanism causing the disease: affected PWS cases inherit two copies of the imprinted maternal 15q region (maternal UPD) with no copies of the non-imprinted paternal genes while in affected AS cases, there are two copies of the imprinted paternal 15q region (paternal UPD) with absence of the non-imprinted maternal genes [11]. Imprinting centre anomalies (deletions, point mutations) account for around 1-3% of cases with PWS [1] and 2-5% of AS cases [2]. Around 10 % AS cases have mutations of the UBE3A gene located within 15q11.3 [2, 13].
PWS patients with UPD have higher verbal IQs, milder behaviour problems and a higher risk of psychosis and autistic spectrum disorders [3]. Among AS cases with UPD, better physical growth, psychomotor development, language ability, and fewer movement abnormalities and ataxia and a lower prevalence of seizures are observed than do those with other underlying molecular mechanisms such as deletion and imprinting centre defects [14]. Deletion cases have hypopigmentation related to loss of the P gene on chromosome 15 [15].
Genetic testing of clinically suspected cases is recommended as it enables management strategies to be targeted early for affected cases including strict dietary control and growth hormone therapy for PWS and alternative communication strategies for AS. The first line investigation to confirm the diagnosis of PWS or AS is the detection of the abnormal pattern of methylation of chromosome 15q11-13 [16, 17]. Methods used include methylation specific polymerase chain reaction (MS-PCR), which involves detection of the methylation pattern of the SNRPN gene [18], which is paternally expressed with the maternal copy being methylated. The investigation involves bisulfite treatment of DNA where the cytosine residues of unmethylated DNA is converted to uracil (the methylated allele is resistant to this change) followed by PCR using primers specific for the differentially methylated sites within the SNRPN region [18] Methylation specific multiple ligation dependent probe amplification (MS-MLPA) utilizes a one-step approach using multiple specific probes around the chromosome 15q region and methylation specific restriction enzyme (Hha1) [19]. Droplet digital PCR involves bisulfite reduction followed by PCR utilizing a sensitive, digital capture methods [20, 21].
Both deletion and duplication cases have low sibling recurrence risks and confirmation of the disease and identification of its mechanism enables more accurate genetic counselling. Methods used to detect the deletion include fluorescence in situ hybridization (FISH) and chromosome microarray (CMA). Family studies using linked microsatellite repeats are able to differentiate between deletion and UPD and determine the parent of origin. Whole exome sequencing has identified 15q11-13 deletions as well as indicate presence of uniparental disomy [20], diagnose unsuspected autosomal recessive mutations of non-imprinted genes located in 15q11-13 as well as other, associated diseases. In rare cases, when PWS/ AS is caused by a parental balanced chromosome translocation, karyotyping is required. In cases fulfilling the diagnostic criteria for PWS and AS, and are negative for the conventional methylation based investigations, imprinting centre anomalies (deletion, point mutation or methylation anomaly) will need to be investigated [20, 22]. Around 10% of patients with AS have a point mutation involving UBE3A and sequencing of this gene needs to be performed [13].
Sri Lanka is an Indian Ocean island with a population of nearly 22 million. It is a middle income country (per capita income $ US 4060-world bank 2019) with wide income disparity [23]. It has significantly reduced infant and childhood mortality and morbidity related to common, preventable infectious disease and genetic diseases are now an important contributor to these indices. Sri Lanka has a health service that is free at the point of delivery but genetic testing is of limited availability and is mostly paid for by the parents of affected children. The availability of low cost testing is essential to enable access to genetic testing as currently, most diagnoses are based on clinical criteria alone.
We report the development of a methylation specific PCR [18] and microsatellite assay to identify affected cases of PWS and AS among Sri Lankan patients. These finding are of relevance to other low and middle income countries attempting to deliver genetic diagnostic services.