Weaver syndrome (MIM #277590) is a rare condition caused by constitutional mutations in EZH2 gene which were described in 1974 by Weaver et al. [1]. This classical overgrowth syndrome manifests by increased mass of multiple tissues, prenatal and postnatal overgrowth, often include advanced bone age, facial dysmorphism including hypertelorism, almond-shaped palpebral fissures, a broad forehead, long ears, retrognathia and a pointed, “stuck-on” chin with horizontal skin crease as well as developmental delay and intellectual disability [2]. Recently Cohen et al. identified previously undescribed de novo mutations in EZH2’s partner protein EED in 2 patients with Weaver-like syndromes [3, 4]. Furthermore, two new patients with the Weaver-like overgrowth syndrome with EED mutation were described by Cooney et al. and by Imagawa et al. [2, 5]. In this report, we present a patient with a clinical diagnosis of Weaver syndrome associated with novel de novo variants in EED.

The proband was the second male child of healthy non-consanguineous parents, family history was non-contributory. The boy was born at 38 weeks of gestation by C-section after an uneventful pregnancy with the birth parameters: weight 3550 g (50th percentile), length 54 cm (25–50th percentile), occipito-frontal circumference (OFC) 34 cm (25th percentile), Apgar score 7 points at 1 min. Bilateral cryptorchidism was noticed. Psychomotor development was delayed (siting at 11 month of age, walking at 2 years, first words at 4 years) but somatic development was accelerated. There was bilateral hypoacusis (50 dB). Retrospectively, based on family photos facial features in the infant period were evaluated showing typical features of Weaver syndrome (Fig. 1). Moreover, advanced bone age on the X-ray of the hand was noted (at the age of 6 years, the bone age was 9 years). MRI of brain showed slightly non-specific enlargement of both ventricles. EEG exam was normal.

Fig. 1
figure 1

a Proband’s facial features in the infant period. Note the typical features of Weaver syndrome with prominent chin. b, c Proband’s facial features at the age of 8 (Color figure online)

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On the current physical examination at the age of 8 years, body weight was 46.6 kg (3 kg above 97 percentile), height 154 cm (14 cm above 97 percentile), OFC 55 cm (97 percentile). The child is hypertrophic and hypotonic. The walk is clumsy, scoliosis is observed. Proband has characteristic facial dysmorphic features: fine hair, wide and prominent forehead, large ears, widely spaced eyes, epicanthic folds, almond shaped palpebral fissures, prominent and deep philtrum, open mouth, widely spaced teeth, retrognathia, and a pointed chin (Fig. 1). The hand and feet are large with hyper-mobile fingers. The club and flat feet, and hoarse voice were noted. Speech development was delayed and moderate intellectual disability with friendly personality was observed. There is no history of cancer suspicion in the proband.

After clinical suggestion of Weaver syndrome, EZH2 gene was sequenced without any pathogenic mutations. Afterward, WES study was performed using SureSelectXT Human All Exon V5 (Agilent) on HiSeq 1500 (Illumina). The mean depth of coverage was 89×, 95% of target sequence was covered min 20× and 99% min 10×.

Bioinformatics analysis of WES was performed as described previously [6] (see also Supplementary Material, Table S1, Fig. S1). It revealed 4 potential pathologic variants: one in ARHGAP35, another in HERC1 and two in EED gene (the details of the filtering process are given in supplement). Family study using amplicon deep sequencing (ADS, performed with Nextera XT Library Preparation Kit) showed that the heterozygous ARHGAP35 variant was inherited from mother, the heterozygous HERC1 variant was inherited from father whereas the two heterozygous variants, c.917 G>C (p.Arg306Thr) and c.919 A>G (p.Asn307Asp) in the EED gene (NM_003797.3) were absent from parents indicating they were de novo mutations. Inspection of individual NGS reads indicated that these EED variants were located in the same allele (in cis, Fig. 2). These variants in proband were further confirmed as de novo mutations using Sanger sequencing (Fig. 2). ADS also confirmed the in cis configuration (Fig. S2). Given current nomenclature recommendations (http://varnomen.hgvs.org) the detected EED variants are described as c.917_919delinsCGG/p.(Arg306_Asn307delinsThrAsp). Both aminoacid changes in the EED gene are likely to be damaging with CADD score = 33 for p.Arg306Thr and 25.5 for p.Asn307Asp, moreover pathogenicity estimation by Polyphen2 and MutationTaster predicted both mutations as deleterious. Both variants are absent from ExAC, gnomAD and our in-house database of >500 Polish exomes. Paternity was confirmed analyzing 17 hypervariable STRs (AmpFLSTR™ NGM SElect™ PCR Amplification Kit, ThermoFisher Scientific, odds in favor of paternity > 106).

Fig. 2
figure 2

Genetic results. a WES results regarding EED gene in proband c.917_919delinsCGG/p.(Arg306_Asn307delinsThrAsp)—view from Integrative Genomic Viewer (IGV); b Sanger sequencing results for proband and his parents—view from Variant Reporter 1.1; c Schematic presentation of known and novel variants in the EED gene. Coding exons are marked with blue rectangles, whereas green rectangles represent non-coding UTRs. Seven WD repeats are marked as yellow rectangles. Mutations described in this report are in red (Color figure online)

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Discussion

We have ascertained an 8 y old boy with clinical symptoms of overgrowth and Weaver syndrome suspicion, in whom two novel mutations affecting two neighboring aminoacids encoded by one allele of the EED gene were found: c.917_919delinsCGG/p.(Arg306_Asn307delinsThrAsp).

In WS patients with EED mutations the following clinical features were observed: abnormal brain MRI findings (substantial white matter volume loss and moderate to severe thinning of the corpus callosum in one patient), genito-urinary anomalies (2 patients), heart defect (ASD and PDA in 2 patients), umbilical hernia (1 patient), ophthalmological abnormalities (myopia, exotropia, 3 patients), skeletal abnormalities (scoliosis, club foot) as well as hearing loss (1 patient), skin and ligament abnormalities, and rough low voice (Table 1) [2,3,4,5].

Table 1 Comparison of patients with EED mutation described in literature with our proband
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Patients described in literature with WS phenotype and EED mutations don’t present leukemia nor embryonic cancers, but the number of patients is still small. In contrast patients with WS caused by mutations in EZH2 suffer from cancers like AML, ALL, neuroblastoma, lymphoma, sacrococcygeal teratoma [7]. Long-term observation and a bigger group of patients with WS-like phenotype and EED mutation are needed to determine if there is a higher risk of cancer among these patients.

In conclusion, we present a patient with a clinical diagnosis of Weaver syndrome and novel de novo sequence variant in EED. Our observation together with previous reports [2, 3, 5] suggests that EED gene testing is warranted in patients with the overgrowth syndrome features and suspicion of Weaver syndrome with normal results for EZH2 gene sequencing. Additional patients with pathogenic variants in EED gene should be systematically evaluated for better understanding the phenotype and the differences between patients with EZH2 and EED gene mutation.