Experimental research
Methylation analysis in tongue tissue of BWS patients identifies the (EPI)genetic cause in 3 patients with normal methylation levels in blood

https://doi.org/10.1016/j.ejmg.2014.03.011Get rights and content

Abstract

The Beckwith–Wiedemann syndrome is caused by disturbed imprinting of genes at 11p15.5. Routine diagnostic testing for Beckwith–Wiedemann syndrome (BWS) includes methylation analysis of the imprinting centers ICR1 and ICR2 in DNA extracted from lymphocytes. In approximately 15% of BWS patients the diagnosis cannot be molecularly confirmed. In this study we determined the methylation status in resected tongue tissue of 11 BWS patients and compared this to the genetic defects found by routine diagnostic screening of blood lymphocytes. In all three patients with normal methylation levels in blood, aberrant methylation patterns were found in tongue tissue. In two patients a UPD was detected and the third case had hypermethylation of ICR1. This result shows that tissue specific mosaic (epi)genetic changes, not present in blood, is the underlying defect in at least a subset of BWS patients without a molecular diagnosis after standard genetic testing.

Introduction

Beckwith–Wiedemann Syndrome (BWS) is a pediatric overgrowth disorder mostly diagnosed at birth, or during early childhood. The natural history of BWS can include prematurity due to macrosomia and polyhydramnion and one third to one half of the affected pediatric patients are reported to have neonatal hypoglycemia [Pettenati et al., 1986, Engstrom et al., 1988], possibly caused by Islet cell hyperplasia and hyperinsulism. The most common features of BWS are macroglossia, which is seen in 80–99% of patients with BWS, followed by pre-or postnatal overgrowth (growth > 90th centile) in ∼90% and anterior abdominal wall defects (exomphalos, umbilical hernia, or diastasis recti) in ∼80 percent [Cohen, 2005, Elliott et al., 1994, Engstrom et al., 1988, Martinez and Martinez, 1996, Pettenati et al., 1986]. BWS patients have an increased risk to develop childhood tumors most commonly Wilms tumor, hepatoblastoma and adrenal cortical carcinoma [Rump et al., 2005]. Overall tumor risks vary from 3% to 43% in infants and young children diagnosed with BWS, and is dependent on the genetic cause of BWS [Bliek et al., 2001].

BWS is caused by disturbed imprinting at chromosome 11p15. The chromosome 11p15 region harbors two independently regulated clusters of imprinted genes. The first cluster contains the reciprocally imprinted genes IGF2 and H19 and is under the control of imprinting centre 1 (ICR1) upstream of the H19 promoter. This ICR is differentially methylated and methylation is present only at the paternal allele. The second cluster contains, among others, the maternally expressed CDKN1C gene and the paternally expressed KCNQ1OT1 gene and is under the control of imprinting centre 2 (ICR2), located upstream of the KCNQ1OT1 promoter. This region is methylated on the maternal allele only [Lee et al., 1999].

In the majority of patients (80%), the syndrome is caused by aberrant imprinting in either one or both imprinted clusters [Bliek et al., 2001, Weksberg et al., 2005]. Aberrant methylation of both ICRs is usually due to a paternal uniparental disomy (UPD) of the 11p15 region (20% of BWS cases) [Bliek et al., 2001]. In a small percentage of patients BWS is caused by mutations in the CDKN1C gene [Lam et al., 1999]. The UPD and imprinting defects in BWS are very often mosaic, indicating that it results from a postzygotic event. Due to this mosaic nature the levels of UPD cells or aberrantly methylated cells may differ per tissue. In this study we determined the methylation status in resected tongue tissue of 11 BWS patients and compared this to the genetic defects found in blood. We were able to confirm the clinical diagnosis in tongue tissue of three patients that had normal methylation levels upon routine diagnostic testing done on DNA extracted from blood lymphocytes.

Tongue tissue from 11 patients was available for analysis. There were 6 males and 5 females. Routine DNA diagnostic testing on blood samples had resulted in the following molecular genetic subtype distribution: aberrant imprinting of ICR2 in 5 patients, aberrant imprinting of ICR1 in 2 patients, UPD in 1 patient and normal methylation levels were found in 3 patients. In all cases, the tongue reduction surgery was performed using the Anterior, V-shaped, Wedge Resection (AWR) technique [Kadouch et al., 2012]. A sample of the excised lingual tissue was obtained for genetic analysis. Tongue biopsies of 8 deceased persons were taken as controls (age range 55–88 yrs).

All patients had the clinical diagnosis of BWS according to the criteria defined by DeBaun and Tucker (1998). The three patients (cases 9–11) with normal methylation levels in blood are described in more detail.

Case 9. A 1.5 year-old boy was admitted to the department of pediatrics of our institution. He was born after 36 and half weeks of gestation with a birth weight of 3130 g (+1SD). Both parents were healthy and there was no family history of medical disorders. During pregnancy, polyhydramnion was observed as well as an adrenal cyst that disappeared afterward. After birth, an exomphalos was detected. Further examination revealed macroglossia, and a low blood glucose level, confirming the diagnosis Beckwith–Wiedemann syndrome (Fig. 1a,b). At presentation, his body length, weight and head circumference all were normal. Both arms were equal in length and size, however an asymmetry was observed in length and size of the legs and feet (right > left). Other symptoms such as naevus flammeus and ear creases or pits were not found. Chromosomal analysis revealed a normal male karyotype and molecular analysis showed normal methylation levels of ICR1 and ICR2 in DNA isolated from blood. After evaluation of the macroglossia by the multidisciplinary craniofacial unit, a partial glossectomy was performed.

Case 10. A 3 month-old boy suffering from macroglossia was presented at our Craniofacial Unit of our institution. He was born after 39 1/7 weeks of gestation with a birth weight of 3775 g (+1SD). Both parents were healthy and there was no family history of medical disorders. Severe macroglossia was detected immediately after birth, the right side being larger than the left side of the tongue. Further examination revealed a cardiac murmur and a hernia umibilicalis, confirming the diagnosis of Beckwith–Wiedemann syndrome (Fig. 1c,d). At two weeks of age, his body length was 52.5 cm (0 SD), weight was 4160 g (+0.5 SD) and head circumference was 38.5 cm (+1.5 SD). Both arms and legs were equal in length and size. Other symptoms such as naevus flammeus and ear creases or pits were not found. Chromosomal analysis revealed a normal male karyotype and molecular analysis showed normal methylation levels of ICR1 and ICR2 in DNA isolated from blood. Tongue reduction surgery was performed at the age of 6 months.

Case 11. A 17 months old boy was seen in the Beckwith outpatient clinic. He was born at 40 weeks of pregnancy with a birth weight of 4740 g (>+2SD). Both parents were healthy and there was no history of medical problems. After birth no hypoglycaemia was present. Physical examination revealed a naevus flammeus, macroglossia, diastasis recti and a right sided hemihypertrophy of arms and legs (Fig. 1e,f) At presentation his length was 84 cm (+1SD), his weight 13.4 kg (+1SD). Molecular analysis showed normal methylation levels of ICR1 and ICR2 in DNA isolated from blood. After evaluation of the macroglossia by the multidisciplinary craniofacial unit, a partial glossectomy was performed at the age of 1 year and 9 months.

Section snippets

Methods

DNA was isolated from blood lymphocytes or buccal swap using Gentra chemicals (Qiagen). The resected tongue tissue was immediately frozen at −20 °C and upon thawing isolated by the same method. Methylation levels were determined by methylation sensitive high resolution melting analysis (MS-HRMA) as previously described [Alders et al., 2009]. UPD analysis was conducted by analysis of STR microsatellite markers in the 11p15 region. PCR amplification of microsatellite markers D11S1288, TH and

Results

Methylation levels of ICR1 and ICR2 in DNA extracted from blood and tongue tissue were determined and compared (Table 1). In patients with aberrant methylation levels in blood the same changes were present in tongue tissue. However, where the methylation levels of ICR1 in tongue were comparable to those found in blood, the methylation levels of ICR2 were relatively higher in tongue than in blood in many patients. In 3 out of 5 cases with ICR2 hypomethylation in blood (cases 1–5) the

Discussion

In the patients with known methylation defects at either ICR1 or ICR2 in blood (cases 1–7), methylation levels at ICR1 in tongue were comparable to those in blood. However, in three patients the methylation levels at ICR2 were higher (less hypomethylated) in tongue than in blood. Since the tongue tissue was severely affected tissue we would have expected that methylation differences, if present, would be more pronounced in this tissue. The hypermethylation of ICR2 in tongue in case 6, who has

Acknowledgments

We are grateful to the patients and their parents for their cooperation.

References (17)

There are more references available in the full text version of this article.

Cited by (25)

  • Causes and Consequences of Multi-Locus Imprinting Disturbances in Humans

    2016, Trends in Genetics
    Citation Excerpt :

    Following this study, several groups confirmed MLID in BWS cohorts, with a frequency of up to 30% of those individuals with an underlying KCNQ1OT1 methylation defect [27,28,35–40]. Two recent papers described methylation anomalies at additional imprinted loci in patients with H19 hypermethylation [41,42]. The MLID observed in BWS are notably different from those observed in TNDM, with both gains and losses of methylation observed at maternal and paternal DMRs.

  • Congenital imprinting disorders: Application of multilocus and high throughput methods to decipher new pathomechanisms and improve their management

    2015, Molecular and Cellular Probes
    Citation Excerpt :

    This is particularly recognized for epimutations in SRS and BWS. The level of mosaicism shows a broad range, and can differ remarkably between different tissues [15,16]. Therefore, if the tissue source of the diagnostic DNA sample has a level of mosaicism below the sensitivity of the diagnostic test, the result will be negative and the patient will escape diagnosis.

View all citing articles on Scopus
View full text