Description of the molecular and phenotypic spectrum of Wiedemann-Steiner syndrome in Chinese patients

Wiedemann–Steiner syndrome (WDSTS, OMIM #605130) is a pleiotropic and extremely rare autosomal dominant disorder, which was first reported by Wiedemann et al. in 1989 and later described by Steiner et al. in 2000 [1]. Using whole-exome sequencing (WES), Jones et al. revealed that germline heterozygous variations of the KMT2A gene (OMIM #159555) was responsible for WDSTS in five patients in 2012 [2]. The KMT2A gene, also known as MLL, encodes a histone lysine methyltransferase that plays a critical role in regulating gene expression during early development and hematopoiesis [3]. Because KMT2A regulates multiple Hox and Wnt genes through histone H3 lysine 4 (H3K4) methylation [4], phenotypes of the WDSTS patients are complex and involve multiple systems, including facial features, skeletal development, and neuro development. A recent summary study pointed out that the clinical features of developmental delay (DD), intellectual disability (ID), postnatal growth retardation, palpebral fissures, down-slanted, wide nasal bridge, broad nasal tip, long eyelashes, and thick eyebrows are more common in WDSTS patients [5]. Increasing number of WDSTS patients of different ethnicities are being identified due to the rapid advance of DNA sequencing technologies, which continues to expand the phenotype spectra with novel features [5]. To date, at least 69 WDSTS patients with variations in KMT2A gene were confirmed, of whom only two are Chinese [5‐11]. Though most of the features in the two Chinese patients are similar to patients of other ethnicities, whether the absent palmar proximal transverse creases is a unique feature of Chinese patients and whether there are novel phenotypes in Chinese patients remain unclear. Meanwhile, our knowledge on the phenotype spectrum and variation spectrum of Chinese patients are still very limited.

Here, we studied the phenotypes and KMT2A variations of 14 unrelated Chinese WDSTS patients. We identified 13 de novo heterozygous variants in the KMT2A gene, including 10 truncating, 2 missense, and 1 splicing variants. Of them, 11 are novel. The phenotypes of Chinese WDSTS patients were compared with a French cohort. In addition, we hereby report several novel clinical features of WDSTS, including macrocephaly, deep palmar crease, external ear deformity, carpal epiphyseal growth retardation, dyslipidemia, and glossoptosis.

In this study, we recruited 14 unrelated Chinese WDSTS patients. To our knowledge, this is the largest cohort of Chinese WDSTS patients with the greatest age span (from pediatric to adult) that have been reported. Recently, Sun et al. reported two Chinese male patients who presented with absent palmar proximal transverse creases, which may be a unique characteristic of Chinese WDSTS patients [6]. We thus examined all the 14 patients and found that no patients had abnormal palmar crease except for Patient 9, who had deep palmar crease (Fig. 1c). We speculate that the lack of abnormal palmar crease in patients of other ethnicities may be a result of negligence during phenotype assessment. Additionally, we revealed several novel clinical features in WDSTS patients, including macrocephaly, deep palmar crease, external ear deformity, carpal epiphyseal growth retardation, dyslipidemia, and glossoptosis.

Though it seems that several clinical features have frequency differences in the Chinese and French cohorts, only 3 (ptosis, thick eyebrows, and feeding difficulties) show statistical significance that might due to lack of enough Chinese WDSTS samples. Prior to the French cohort study, delayed bone age had been reported in at least 3 patients [6, 17], while significantly advanced bone age was first and only described in a 4-year-old female patient [18]. Here, the French cohort and our cohort reported another 9 and 12 patients presented with advanced bone age and delayed bone age, respectively. Therefore, we propose that both of advanced bone age and delayed bone age are authentic phenotype of WDSTS caused by KMT2A variation. Patients 3, 4 and 10 had GH deficiency and also presented with postnatal growth retardation, which are consistent with the results from the French cohort. Moreover, maternity history of Patient 7 supports the surmise of normal fertility in female WDSTS individuals, which has also been reported in two French patients.

The French cohort reported a healthy unaffected father with very low proportion of mosaicism in the KMT2A gene (p.Arg1154Trp), who had two affected daughters. Intriguingly, Patient 10 in our cohort was the first WDSTS case reported with mosaicism in the KMT2A gene (c.5871 T > A; p.Tyr1957*), who on the other hand presented with more severe phenotypes as well as several previously unreported symptoms (external ear deformity, carpal epiphyseal growth retardation, and dyslipidemia).

Because WDSTS can phenotypic overlap with Pierpont syndrome, Cornelia De Lange syndrome and Kabuki syndrome [6, 7, 9, 19, 20], the candidate genes causing the later three syndromes (i.e. TBL1XR1, NIPBL, SMC1A, SMC3, RAD21, HDAC8, KMT2D, and KMD6A) in our cohort have been excluded. To date, a total of 72 KMT2A variants spanning the whole protein have been identified (including the variants identified in this study), of which 71 are point variants and only one is intragenic large deletion [5‐11]. As shown in Additional file 2: Table S2, of the 71 point variants, 48 are truncating, 17 are missense, and 6 are splicing variants. The p.Ser774Valfs*12 variant in Patients 3 and 7 has been previously reported in 3 unrelated patients [11, 21], which qualifies Ser774 as a hotspot for gene variation in KMT2A. Other 5 recurrent variants, including p.Arg1154Trp [11], p.Cys1155Tyr [11, 22], p.Gly1168Asp [14, this study], p.Arg1633* [2, 11] and p.Arg2480* [7, 11] were also identified. Although we did not find hotspot variation region, it is noteworthy that only missense variants (7/17) occurred in the cysteine-rich CXXC zinc finger domain (Additional file 2: Table S2), which is predicted to selectively bind to unmethylated CpG-containing stretches of the target genes [10]. Previous studies suggested that patients who harbor the missense variant in the CXXC DNA binding domain may show more severe neurodevelopmental delay [10, 14]. Indeed, in our cohort, Patient 5 (p.Gly1168Asp) has the most severe anomalies in neurological development. In contrast, Patient 4, who has a missense variant (p.Arg3906Cys) in the SET domain showed less neurological abnormalities, although the SET domain plays an important role in transcriptional activation via its H3K4 methyltransferase activity [5]. In the French cohort, 5 patients (Patients 24–28) harbored the missense variants in the CXXC DNA binding domain. 3 of them showed severe ID, while only one patient with severe ID was observed in the other 28 patients. 4 of them had persistent hypotonia (8 in the other 25 patients) and 3 of them had seizures (only one in the other 26 patients). These results further and firmly demonstrate the relationship between dysfunction in the CXXC DNA binding domain and severe nuerophenotypes.

Most of the KMT2A variants generate truncated products (the same in Chinese patients, 12/16) that further suggest haploinsufficiency is the main pathogenesis of KMT2A gene. It is speculated that missense variants of the KMT2A gene are more likely to cause WDSTS by a dominant negative effect [23]. The hypothesis is supported by the fact that upregulation of the KMT2A gene transcription in patient with p.Arg1154Trp and site-specific DNA methylation changes driven by KMT2A missense variants [9, 10], which might explain the missense variants in the CXXC DNA binding domain linked to a more severe neurophenotypes. Nevertheless, more functional study need be performed to figure out the molecular mechanism, especially why there were only missense variants in this domain.

Currently, it is still difficult to have more conclusions of genotype and phenotype in WDSTS patients due to its broad spectrum of phenotypes. Further studies should attempt elucidating the connection between genotype and phenotype, with epigenotype in consideration. In addition, the clinical interventions for WDSTS patients are yet limited. For most patients, symptomatic treatment, such as rehabilitation training, is the only option to improve the motor development. Results from Patient 3 in our study and another reported 2 patients [6, 16] indicate that GH therapy may be an effective method to improve the height of patients with GH deficiency. Of course, clinical safety should be taken into consideration when using growth hormone therapy.

In summary, we report 14 Chinese WDSTS patients with 13 pathogenic variants in the KMT2A gene, including 11 novel variants. Our detailed comparison between the Chinese and French patients indicates no significant difference in phenotypic spectrum; however, the frequency of several symptoms were different. We demonstrated that variation in the KMT2A gene can lead to both advanced and delayed bone age. The novel phenotypes, including macrocephaly, deep palmar crease, external ear deformity, carpal epiphyseal growth retardation, dyslipidemia, and glossoptosis further extended the WDSTS phenotype spectrum. Our study supports the notion that the CXXC zinc finger domain is a hotspot region for missense variants, which is associated with more severe neurophenotypes. In addition, growth curves and endocrine-related problem need further investigation in the future.