Prader-Willi syndrome: Methylation study or fluorescence in situ hybridization first

Indian Journal of Human Genetics September-December 2010 Volume 16 Issue 3

Address for correspondence: Dr. Sellama Nadifi, Laboratory of Human Genetics, Faculty of Medicine, Casablanca, Morocco.

E-mail: labgenmed2@yahoo.fr

Prader-Willi syndrome: Methylation study or fluorescence in situ hybridization first?

Khalil Hamzi, Afaf Ben Itto

, Sanaa Nassereddine, Sellama Nadifi

Laboratory of Human Genetics, Faculty of Medicine,

Department of Pediatrics, University Hospital, Casablanca, Morocco

Prader–Willi syndrome (PWS) is neurogenetic disorder involving the imprinting mechanism at 15q11–13 region. We report a 4-year-old girl who was referred to our laboratory to be investigated for clinical obesity, mental deficiency and respiratory problems. The patient was born for non- consanguineous and healthy biological parents. After normal pregnancy, the patient was delivered by cesarean section at full term, with a birth weight of 2500 g, and the height and head circumference were unknown. In neonatal stage, she presented severe hypotonia with feeding problems. Her developmental progress was delayed. She walked and developed speech at the age of 3 years. Since the age of 3 years, she presented severe dental problems.

Methylation study had confirmed the diagnosis, and for detecting etiology, fluorescence in situ hybridization using probes for small nuclear ribonucleoprotein polypeptide N (SNRPN), which map inside the chromosomal region 15q11–15q13, was necessary to confirm the 15q11–

15q13 deletion of paternal chromosome 15, which is the predominant genetic defect in PWS. In conclusion, we report this case with an objective to reinforce the necessity of analysis of DNA methylation within the 15q11–13 region, which is an important tool for the correct diagnosis among children presenting with neonatal hypotonia, mental deficiency and obesity.

Key words: Fluorescence in situ hybridization, methylation, obesity, Prader–Willi syndrome

Case Report

or early childhood by excessive eating and gradual development of morbid obesity. Approximately 70% of individuals with PWS have a 15q11.2–q13 deletion on the paternally inherited chromosome 15, whereas 25%

have maternal uniparental disomy (UPD), 5% have an imprinting center sequence variant and 1% of the patients have a structural chromosome rearrangement involving 15q11.2–q13. Regardless of the etiology, PWS patients obtain only the methylated allele(s) in the promoter region of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene.

Case Report

We report a 4-year-old girl who was referred to our laboratory to be investigated for clinical obesity, mental deficiency and respiratory problems. The patient was born for non-consanguineous and healthy biological parents. After normal pregnancy, the patient was delivered by cesarean section at full term, with a birth weight of 2500 g; height and head circumference were unknown. In neonatal stage, she presented severe hypotonia with feeding problems. Her developmental progress was delayed. She walked and developed speech when she was 3 years old. When she was examined at the age of 3 years, she presented severe dental problems, mental retardation and a weight of 50 kg for 100 cm [Figure 1].

Methylation study using allele specific primers has confirmed the diagnosis of PWS showing the absence of the non-methylated paternal copy of SNRPN [Figure 2].

DOI: 10.4103/0971-6866.73417

Introduction

Prader–Willi syndrome (PWS; OMIM 176270) is one of the most common genetic disorders involving non- Mendelian inheritance in the form of genomic imprinting.

It occurs with an incidence of 1 in every 15,000 live births

and is characterized by severe hypotonia and feeding

difficulties in early infancy, followed in later infancy

173

Figure 1: (a) Show the typical dysmorphic features of Prader Willi. (b) Severe dental problems. (c) Figures showing the severity of obesity (50 kgs for 4 years old). (d) Figures showing the severity of obesity (50 kgs for 4 years old).

Discussion

Methylation analysis by Southern blotting using SNRPN probe detects over 99% of subjects with PWS.

The SNRPN probe has been validated as a diagnostic test for PWS.

Few subjects have been reported in the literature with typical PWS features and normal methylation studies.

Actually, several methods involving polymerase chain reaction (PCR) analysis for the molecular diagnosis

of PWS have been developed, including methylation specific PCR (MS-PCR), fluorescence melting curve analysis (MS-HRMA) and reverse transcription PCR (RT-PCR).

These techniques have several advantages over Southern blot analysis for the diagnosis of PWS, including a rapid turnaround time and a smaller amount of DNA template required. As with Southern blot analysis, each method detects virtually all cases of PWS but they do not give any information on the etiology of the PWS.

Approximately 70% of patients with PWS carry microdeletions and most of the remaining PWS patients were later found to have UPD for chromosome 15.

The etiologic heterogeneity of PWS makes it difficult to establish uniform diagnostic methods that would detect most positive cases. Fluorescence in situ hybridization (FISH) analysis to identify the large microdeletions on the chromosome 15q is available, but only 70% of PWS Figure 2: PCR Profil obtained with MS-PCR (N=Normal,

PW = Prader Willi).

Hamzi et al. : Prader-Willi syndrome

174

Figure 3: Diagnosis strategy of Prader-Willi syndrome.

cases are identified.

The diagnostic approaches that the different laboratories use differ from each other.

Some laboratories begin their examinations with FISH to detect deletions, followed by the DNA methylation test, if the deletion is not found. However, beginning with the DNA methylation test, deletions as well as uniparental disomy and imprinting defects can be recognized.

In the case of an abnormal methylation imprint, FISH and microsatellite analysis are recommended to distinguish between microdeletion and uniparental disomy [Figure 3].

References

1. Buchholz T, Jackson J, Robson L, Smith A. Evaluation of methylation analysis for diagnostic testing in 258 referrals suspected of Prader-Willi or Angelman syndromes. Hum Genet 1998;103:535-9.

2. Melinda P, Lan-Szu C, Wei T, Mohamed J, Rong M.

Molecular diagnosis of prader–willi and angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification. Clin Chem 2006;52:1276-83.

3. Sun Y, Nicholls RD, Butler MG, Saitoh S, Hainline BE, Palmer CG. Breakage in the SNRPN locus in a balanced 46, XY, t(15;19) Prader-Willi syndrome patient. Hum Mol Genet 1996;5:517-24.

4. Kosaki K, McGinniss MJ, Veraksa AN, McGinnis WJ, Jones KL. Prader-Willi and Angelman syndromes: Diagnosis with a bisulfi te-treated methylationspecifi c PCR method. Am J Med Genet 1997;73:308-13.

5. Ledbetter DH, Engel E. Uniparental disomy in humans:

Development of an imprinting map and its implications for prenatal diagnosis. Hum Mol Genet 1995;4:1757-64.

6. Velinov M, Jenkins EC. PCR-based strategies for the diagnosis of Prader-Willi/Angelman syndromes. Methods Mol Biol 2003;217:209-16.

7. The statement of the American Society of Human Genetics and American College of Medical Genetics 1996.

8. Stalker HJ, Williams CA. Genetic counseling in Angelman syndrome: The challenges of multiple causes. Am J Med Genet 1998;77:54-9.

Source of Support: Nil. Conflict of Interest: None declared.

Hamzi et al. : Prader-Willi syndrome

Copyright of Indian Journal of Human Genetics is the property of Medknow Publications & Media Pvt. Ltd.

and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright

holder's express written permission. However, users may print, download, or email articles for individual use.