CSM Ng, CWM Au, HM Luk

Abstract

Beckwith-Wiedemann syndrome (BWS) is a genetic disorder characterised by overgrowth and an increased risk of embryonal tumours. Here, we present two local pairs of monozygotic twins with BWS. In both cases, one twin exhibited more severe features while the other twin had milder or no manifestations of the syndrome. Genetic analysis revealed concordant loss of methylation at the ICR2 region of chromosome 11p15 in both peripheral blood and buccal samples of the affected twins. Yet the unaffected twins showed loss of methylation only in peripheral blood, suggesting tissue-specific mosaicism of epigenetically aberrant cells. These cases emphasise the variable expressivity of BWS within monozygotic twin pairs and highlight the caveat of using peripheral blood alone for diagnosis. Further research is necessary to elucidate the underlying mechanisms contributing to this phenotypic variability, and long term data is needed to establish whether tumour surveillance is needed for the clinically unaffected ones in particular.

Introduction

Beckwith-Wiedemann syndrome (BWS, OMIM #130650) is a clinically diverse genetic disorder characterised by somatic overgrowth and associated with an increased risk of embryonal tumours. BWS is diagnosed based on clinical examination findings aided by BWS clinical score, the scoring system consisted of cardinal features (2 points each) and suggestive features (1 point each). A clinical diagnosis of BWS can be made if the score is >=4.¹ BWS is mainly caused by genetic or epigenetic defects within the 11p15.5 region containing imprinted genes such as CDKN1C or IGF2, which are regulators of fetal growth. The prevalence is estimated to be 1 in 10340 livebirths.² The commonest causative alteration for BWS is the loss of methylation of imprinting control region 2 (ICR2-LOM), which accounts for 50% of cases. Other causes include paternal uniparental disomy 11 (upd(11)pat), gain of methylation of ICR1 (ICR1-GOM), loss-of-function variants of the CDKN1C gene and paternal duplication of 11p15.¹

Multiple pregnancies increase the incidence of imprinting disorders, whilst the incidence of female monozygotic twins among BWS patients is substantially higher than in the general population. However, the twins usually exhibit different clinical features with one twin being more severely affected than the other. Here, we illustrate two pairs of phenotypically discordant monozygotic twins with BWS.

Case Presentation

Case 1
This was a naturally conceived female monozygotic, monochorionic-diamniotic (MCDA) twin pregnancy. Their 37-year-old father and 33-year-old primigravida mother are Chinese and non-consanguineous, with no family history of BWS. At 22 weeks of gestation, twin-twin transfusion syndrome was suspected due to liquor volume discrepancy. Twin 1 had polyhydramnios and twin 2 had normal liquor volume, there was no discrepancy in the estimated foetal weight. Weekly ultrasound monitoring showed gradual decrease in liquor discrepancy and no intervention was required. The discrepancy resolved at 30 weeks of gestation. The babies were delivered via emergency Caesarean section for preterm labour and prolonged second stage of labour at 35 weeks and 2 days of gestation. Twin 1 weighed 2350 g and twin 2 weighed 2035 g, both appropriate for gestational age. Placental histology post-delivery showed a 3 cm chorangioma and funisitis in twin 1, while the placenta of twin 2 was unremarkable.

Twin 1 was born flaccid without spontaneous breathing at birth, requiring intubation and chest compression in view of bradycardia. She experienced transient hypoglycaemia at 1 hour of life and was stabilised with dextrose infusion. She was also diagnosed to have moderate to severe hypoxic-ischaemic encephalopathy with neonatal seizure and started on intravenous phenobarbital which gradually tailed off. Physical examination revealed a slightly large tongue and right preauricular sinus. She was discharged at 2 months of life with growth parameters at the 50th to 75th centile. The growth parameters had further crossed the centiles and reached 97th centile in both the body weight and body height since 8 months old. She was referred to genetics clinic due to coarse facial features and protruding tongue at 2 years old. She had normal development except mild speech delay, examination showed macroglossia, right preauricular sinus, ear crease, naevus flammeus and umbilical hernia (Figure 1). There was no hemihypertrophy. Ultrasound abdomen showed hepatomegaly. Clinical diagnosis of BWS was substantiated.

Figure 1 Clinical Photos of Twin 1 in Case 1 (with consent for publication). Facial profile of Twin 1 showing features of Beckwith-Wiedemann syndrome including macroglossia, naevus flammeus and horizontal crease over right ear. Other features include right pre-auricular sinus, anteverted nostrils and coarsening of facial features.

Twin 2 was also born flaccid and cyanotic, requiring intubation and chest compression at birth. She had transient hypoglycaemia down to 2.3 mmol/L at 1 hour of life, which normalised after dextrose infusion. She was discharged at 3 weeks of life. She had a 2 cm haemangioma at the right forearm, with no other BWS features observed. Growth parameters were at 75th to 90th centile all along. The Beckwith-Wiedemann spectrum (BWSp) score for twin 1 and twin 2 were 8 and 1 respectively (Table 1).

Further molecular testing was arranged. Zygosity was confirmed by microsatellite testing by AmpFLSTRTM Identifiler^TM kit with DNA of the twin siblings. Peripheral blood for methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) analysis for BWS for both twins showed concordant loss of methylation at ICR2 region of chromosome 11p15 imprinted region. MS-MLPA performed on DNA extracted from the buccal mucosa confirmed the same loss of methylation at ICR2 region in twin 1, but there was no copy number change or abnormal methylation in twin 2.

Case 2
This was a female monozygotic twin with spontaneous conception. This was the first pregnancy of the 34-year-old father and the 30-year-old mother. There was no known family history of BWS. Antenatal course was unremarkable reportedly. The twins were delivered via Caesarean section at 37 weeks of gestation in China. Twin 1 weighed 3400 g at birth with normal development. The details of the delivery record and newborn examination were not retrievable.

Twin 1 was later referred to genetics unit at 2 years old for hemihypertrophy. She was noted to have leg length discrepancy since 6 months of age and frequent tongue protrusion. All growth parameters including head circumference, body weight and body height were at 97th centile. Examination showed naevus flammeus, ear crease, ear pits, right hemihypertrophy and umbilical hernia. Abdominal ultrasound showed no organomegaly. Twin 2 exhibited no features of BWS. The BWSp score for twin 1 and twin 2 were 7 and 0 respectively (Table 1).

Similarly, peripheral blood and buccal swabs were taken for MS-MLPA. Twin 1 showed loss of methylation in ICR2 region of chromosome 11p15 imprinted region in both samples, whilst twin 2 showed loss of methylation at the same region in peripheral blood but not the buccal sample (Figure 2). Zygosity was again confirmed by microsatellite testing.

Figure 2 MS-MLPA Analysis for Case 2. Representative plots of MS-MLPA analysis by Coffalyser using blood and buccal samples from both Twin 1 and Twin 2 (Case 2). Copy number analysis (a,c,e,g) were normal using blood and buccal samples for both twins. On the other hand, methylation analysis (b,d,f,h) showed loss-of-methylation of ICR2 (framed area) in both samples from Twin 1, compared to presence of LOM only in blood sample but not buccal sample from Twin 2. Similar findings were noted for Case 1.

Discussion

These two cases illustrated the variability in degree of phenotypic discordance among BWS multiple pregnancies. The majority of BWS twins are phenotypically discordant, and it has been reported that the average BWSp scores of MCDA twins were 7.8 +/- 0.65 (proband with BWS) and 2.8+/-0.58 (unaffected twin).³ The findings correlated with the two pairs of twins reported in our case report. These two pairs of monozygotic twins showed ICR2-LOM in the peripheral blood leukocytes (PBL) in both the affected and unaffected twin, but discordant results from the DNA sample extracted from the buccal swabs.

We described two pairs of female monozygotic twins. Monozygotic twins with BWS have a higher female-to-male ratio compared to normal twins (0.496-0.512).^4,5 Sex-dependent differences in early embryonic development, currently under debate, have been hypothesised as the underlying mechanism. Male embryos have been observed to reach the final blastocyst stages faster than females, although conflicting findings exist.⁶ Such difference may be attributed to potential mitogenic properties of the SRY gene, dose-dependent mRNA transcription levels due to earlier X-linked gene expression than autosomal gene expression, or varying metabolic activity between the sexes. The delayed embryonic development in females may lead to a failure of methylation maintenance, potentially caused by X-inactivation or abnormal DNMT1o expression.⁴ The failure in epigenetic maintenance in one cell may subsequently serve as a triggering event for twinning.⁷

Previous study speculated the finding of ICR2-LOM in the PBL of the unaffected twin was due to shared blood circulation in the placenta anastomosis.⁵ However, later study found that a common source of haemopoietic stem cells exhibiting ICR2-LOM is more likely the cause.⁴ As loss of methylation occurs before twinning, the mosaic distribution of ICR2-LOM cells in different tissues may result in the phenotypical discordance observed in twins.^3,5 The degree of BWS affectedness and the degree of mosaicism are likely associated to the timing of embryologic twinning in relationship to the timing of epigenetic aberration.³ Buccal swabs were performed in both pairs of twins as it is proposed to give a more reliable methylation due to confounder of shared placental circulation.⁴ On the other hand, there is also case report on dichorionic diamniotic monozygotic twins with BWS with concordant BWSp scores but discordant epigenetic alteration on PBL.⁸ This signifies tissue-specific analysis should be considered the gold standard for the detection of altered methylation.⁹

Different molecular subtypes of BWS show genotype-phenotype correlations in tumour development, with the ICR2-LOM group having the lowest tumour risk but notable variability (2.6%). Recommendations regarding tumour screening for this subgroup have been conflicting - uniform screening regardless of the epigenetic changes is suggested in the United States¹⁰ but not in Europe¹ due to different screening thresholds. Whether tumour surveillance should be initiated for the phenotypically unaffected twin is even more controversial. One previous study advocated a conservative approach in conducting BWS tumour surveillance in the clinically less affected twin, as it is difficult to predict the burden of epigenetic aberration on the associated tumour risk and degree of organ involvement.³

In conclusion, these two pairs of monozygotic twins with BWS provides supporting evidence for the occurrence of ICR2-LOM before twinning, and the mosaic distribution of epigenetically aberrant cells in different tissues as confirmed by the molecular testing in the PBL and buccal swabs. Our case reports illustrated the algorithm in clinically evaluating the twins and the proposed molecular testing and analysis in different tissue types. Continued follow-up data on any tumour formation in this group of children can provide guidance on the recommendations of surveillance.

Declaration of Interest

All authors have indicated that they have no conflicts of interest.

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