Three de novo variants in KMT2A (MLL) identified by whole exome sequencing in patients with Wiedemann–Steiner syndrome

Abstract Background Wiedemann–Steiner syndrome (WSS) is an autosomal dominant disorder characterized by short stature, hypertrichosis, intellectual disability, developmental delay, along with facial dysmorphism. WSS patients exhibit great phenotypic heterogeneities. Some variants in KMT2A (MLL) gene have been identified as the cause of WSS. Methods Whole exome sequencing on the probands followed by Sanger sequencing validations in the family were applied to determine genetic variants. In silico analyses were used for predicting potential effects of the variants. Results We identified three novel de novo heterozygous variants: c.883A>T (p.Lys295*), c.4171C>T (p.Gln1391*), and c.3499T>C (p.Cys1167Arg), in KMT2A gene from three unrelated Chinese WSS patients. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, these three variants were classified as pathogenic, pathogenic and likely pathogenic variant, respectively. By reviewing all the available cases with same mutated KMT2A regions as the three patients had, we found that in addition to the representative symptoms, our patients exhibited some sporadically observed symptoms, such as severe ophthalmological symptoms, endocardial fibroelastosis, cytomegalovirus infection, and feet eversion. We also revealed that variants in different KMT2A regions contribute to the phenotypic heterogeneity of WSS, highlighting challenges in the diagnosis of syndromic disorders spanning a broad phenotypic spectrum. Conclusion Our study would aid in further broadening our knowledge about the genotype–phenotype correlation of WSS.


| INTRODUCTION
Wiedemann-Steiner Syndrome (WSS, OMIM #605130) is a very rare multiple congenital anomaly syndrome that shows autosomal dominant inheritance. WSS is characterized by developmental delay, intellectual disability, short stature, hypertrichosis along with distinctive facial appearance (Jones et al., 2012). The prevalence of WSS is estimated to be less than 1 in 1,000,000 (www.orpha.net, ORPHA: 319182). It was firstly described by Wiedemann et al. (1989). Thereafter, a variety of WSS cases were reported worldwide, but they exhibited great phenotypic heterogeneities (Baer et al., 2018;Grangeia et al., 2020;Jones et al., 2012;Li et al., 2018).
KMT2A encodes a transcriptional coactivator KMT2A (also named MLL), which is crucial for gene expression regulation through its function of histone methyltransferase during early development and hematopoiesis (Ronan et al., 2013). KMT2A is a large protein of 3,969 amino acid residues, and it is a structurally complex protein composed of several conserved functional domains, including three DNA-binding AT-hook at the N terminus, a cysteine-rich CXXC domain, four plant homeodomain (PHD) finger motifs, a bromodomain domain (BD), a transactivation domain (TAD), a FY (phenylalanine tyrosine)-rich N terminus (FYRN) motif, a WDR5 interaction (Win) motif, and finally a catalytic SET (Suppressor of variegation, Enhancer of zeste, Trithorax) domain at the C-terminus.
Among these domains, the SET domain is responsible for histone H3 lysine 4 (H3K4) methyltransferase activity which mediates epigenetic transcriptional activation (Milne et al., 2002). The AT-hook and the CXXC domain are capable of binding AT-rich DNA and nonmethylated CpG-containing DNA, respectively; they are essential for fusing KMT2A to other proteins, such as nuclear transcription factors, transcriptional coactivators, chromatin remodeling protein, as well as cytoplasm heterologous proteins (Liu & Min, 2019;Zeleznik-Le et al., 1994). These fusion proteins are then implicated in various celltransforming processes. The nuclear-targeting sequences located between AT-hook and CXXC domains contribute to the nuclear localization of KMT2A and its fusion proteins (Yano et al., 1997). Previous studies reported that patients with variants in the CXXC domain may display more severe neuro-phenotypes (Lebrun et al., 2018;Li et al., 2018;Min Ko et al., 2017). However, more studies would be needed to further delineate the genotypephenotype correlation of WSS.
In this study, we performed whole exome sequencing on three unrelated Chinese WSS patients, and identified two novel truncating variants and one novel missense variant in KMT2A. We elaborated clinical and genetic features of these cases, and compared them with available WSS cases. We commented on how variants locating in different KMT2A domains contribute to the phenotypic heterogeneity. Our study further expanded the mutation spectrum of KMT2A and provided abundant evidences on the genotype-phenotype correlation of WSS.

| MATERIALS AND METHODS
Our study was approved by the ethics committee and review board of Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital). After obtaining written informed consents from patients' parents, genomic DNA were extracted from the whole blood of three patients and their parents. Trio-based whole exome sequencing was performed on Illumina The first patient was a 10-year-old girl with a history of short stature, growth retardation, intellectual disability, hirsutism, and ocular anomaly. She was born to nonconsanguineous Chinese parents via vaginal delivery at 38 weeks, with a birth weight of 3.1 kg. No obvious intra-uterine growth restriction was observed by antenatal ultrasound scanning. She was noticed to have growth retardation at 1 year of age. At 4 years old, she was referred to our endocrinology department for comprehensive evaluations. Physical exam suggested that she had no obvious facial dysmorphism (Figure 1a left), while her height was 84.8 cm (<−3 SD) and weight was 23 kg (>2 SD). Basal hormonal evaluation revealed normal levels of T3, T4, TSH (thyroid stimulating hormone), CORT (8 am and 8 pm), ACTH (adrenocorticotropic hormone), and IGFBP3 (insulin-like growth factor binding protein), but decreased level of IGF-1 (insulin-like growth factor-1; 33.2 ng/ml, reference range >55 ng/ml), which suggested IGF-1 deficiency. Glucagon stimulation testing showed peak growth hormone (GH) level was 10.5 ng/ ml (reference range >10 ng/ml), indicating no obvious GH deficiency. A hand x-ray showed that her bone age was only 2.5 years. Brain MRI (Magnetic Resonance Imaging) identified increased pituitarium. At the age of 10 years, she came to our rehabilitation department for re-evaluation, since she exhibited symptoms of poor learning ability in mathematics, severe sensory integration dysfunction, as well as defective communication and socialization skills. She presented mild facial asymmetry, epicanthus tarsalis accompanied with severe ptosis of the upper eyelid and small palpebral fissures, as well as serious ocular abnormality of astigmatism (Johnson, 1978). In particular, her height (118 cm) and weight (23 kg) were more than 3 SD below the mean values for her age.
The second patient was a 5-month-old girl with a history of congenital cardiac malformation, recurrent infections, distinctive facial features, global developmental delay, and muscular hypotonia. She was born at the 37th week of gestation by vaginal delivery due to premature rupture of the amniotic fluid, with birth weight of 2.8 kg (<−1 SD) and birth length of 47 cm (<−1 SD). She was the first child of healthy, non-consanguineous parents. Her mother had minor vaginal bleeding and no remarkable history during the prenatal and neonatal periods. At the first month and 22 days, she was referred to our pneumology department due to severe paroxysmal cough and failure to thrive. On examination, her length was 51 cm (<−2 SD), weight 3.5 kg (<−2 SD). She had very low-pitched crying and poor responses to various actions. Cytomegalo virus (CMV) test suggested positive cytomegalovirus infection (CMV-IgG = 3.609 g/L, and CMV-IgM = 7.2243 g/L). Serum immune function testing revealed slightly increased level of IgA (0.36 g/L, reference range 0.14-0.34 g/L) as well as normal levels of C3, C4, IgG, and IgM. Echocardiographic evaluation showed increased left ventricular internal diameter (30 mm, reference range 11.82 ± 1.15 mm for 3-month-old infant; Deng et al., 2006), increased left endocardium, enlarged pulmonary artery, and some left-to-right shunts, indicating the likelihood of endocardial fibroelastosis, patent ductus arteriosus and patent foramen ovale ( Figure 1b). Consequently, she was transferred for cardiac surgery of patent ductus arteriosus ligation at 3.5 months. Re-examination after the surgery did not identify residual shunting at the arterial level. At 5 months, she was referred for re-evaluation due to failure of holding up her head on her own, which was expected by the time of 3 months old. At this age, her height was 59 cm (<−2 SD) and weight 5 kg (<−3 SD). She had malnourished and dysmorphism face, sparse scalp hair, bilateral ptosis, mild hypertelorism, down-slanted palpebral fissure, low-set ears, long eyelashes, long philtrum, hypertrichosis on her arms and legs, thin upper lip, micrognathia, as well as small hands and feet (Figure 1a middle). She was unable to roll over or raise head, and her lower limbs could not bear the body weight when she stood with help. Her developmental assessments on motor skills, learning behaviors, language, and social adaptation using the Gesell Developmental Schedule indicated global developmental delay. Brain MRI showed enlargements in both bilateral fissure and anterior longitudinal division (Figure 1c).

| Patient 3 (P3)
The third patient was a 20-month-old girl with a history of muscular hypotonia, global developmental delay, skeletal anomalies, and unusual face. She was the second child of healthy, non-consanguineous parents, and had a healthy elder brother. Her mother had a remarkable pregnancy history of two spontaneous abortions and two induced abortions. During the prenatal stage of this girl, her mother was diagnosed with an increased level of amniotic fluid volume by ultrasound scanning. She was born via vaginal delivery at 37 weeks of gestation due to premature rupture of the amniotic fluid, with weight of 2.65 kg (<−1 SD) and height of 47 cm (<−1 SD). At birth, she had no distinctive facial dysmorphism, but hirsutism was found on her ears, which fell off after one month. She was able to raise her head at 6 months and sit at 10 months. At 20-month-old, she was yet unable to crawl or walk independently, and referred to the hospital. She had bilateral foot eversion. The neurological examination indicated that four limbs had generalized muscle hypotonia and generalized weakness with Medical Research Council (MRC) grade 4. Her height was 69 cm (<−3 SD) and weight was 10 kg (<−2 SD). She had some distinctive dysmorphic facial features, including prominent forehead, hypertelorism, wide nasal bridge, and low-set ears (Figure 1a, right). Development quotient (DQ) results of her Gesell development examination were 22 in adaption, 27 in gross motor, 22 in fine motor, 31 in speech, and 27 in personal-social, indicating developmental delay. Her cognitive function assessment showed impaired cognitive capacity of only 5-7 months old. The Activity of Daily Living assessment revealed poor activity (score = 7.5). Brain MRI revealed enlargement of bilateral frontal and temporal subarachnoid space, as well as choroid plexus cysts.

| DISCUSSION
We described three patients with neurodevelopment delay together with multiple malformations and identified three novel de novo causative variants in KMT2A, a gene known to be responsible for WSS. In line with WSSrelated reports, the three patients exhibited representative characteristics of WSS, such as short stature, developmental delay, mental retardation, hypertrichosis, and dysmorphic facial features. They also had some other distinctive symptoms which were sporadically observed. Baer et al. reported that eye anomalies were present in 59% of their studied WSS cohort, while the co-occurrence of ptosis and astigmatism were found in only one patient (Baer et al., 2018). Notably, our patient 1 had severe ophthalmological symptoms of epicanthus tarsalis (a type of epicanthic fold), upper eyelid ptosis, small palpebral fissures, and astigmatism. It was reported that 35.44~36% WSS patients had cardiac defects, including 28.89% patent ductus arteriosus, and 8.89% patent foramen ovale (http://www. wssfo undat ion.org/), but WSS case implicated with endocardial fibroelastosis has not been previously reported. In this study, we found that patient 2 presented congenital heart defects of endocardial fibroelastosis, patent ductus arteriosus, and patent foramen ovale. Furthermore, we found that patient 2 had cytomegalovirus infection after birth, which were probably due to prenatal cytomegalovirus infection or congenital immunodeficiency. Although, WSS cases with congenital immunodeficiency had been previously reported, and their main manifestations were recurrent infections of respiratory tract or urinary tract, and significantly reduced immunoglobulin level (IgG, IgA, and IgM) in peripheral blood (Bogaert et al., 2017;Stellacci et al., 2016), patient 2 had normal immunoglobulin and complement levels which were described in detail above. Patient 3 had feet eversion, which has not, to the best of our knowledge, been reported, although puffy or small feet were reported to be in frequency of 22% in WSS cases (Enokizono et al., 2017). Therefore, variants in KMT2A cause a wide spectrum of clinical phenotypes, and WSS is a complex multi-system disorder that might be sometimes difficult for the clinical diagnosis. Endocardial fibroelastosis is a thickening of the endocardium due to layers of collagenous and elastic fibers. It might result from viral infection, inflammation, autoimmunity, metabolic anomalies, developmental defects, and genetic factors (Lurie, 2010). For example, genes (e.g., TAZ, NEX, MYH7, and CSRP3) have been demonstrated to involve the development of endocardial fibroelastosis (Aherrahrou et al., 2016;Brady et al., 2006). Given that our patient 2 had cytomegalovirus infection and genetic defects, and WSS is a multi-systematic anomaly, we suggest that further research on the etiology of endocardial fibroelastosis in WSS cases is necessary in the future.
WSS patients associated with mutated AT-hook (amino acids 169-180, 217-227, and 301-309) of KMT2A are yet limited. p.Lys295* identified in our patient 1 is the fourth variant identified in AT-hook. Two truncating variants p. Glu219Leufs*27 (at AT-hook 2) and p.Arg301* (at AT-hook 3), as well as a missense variant p.Pro280Thr (between AT hook 2 and AT-hook 3) have been previously reported (Baer et al., 2018;Li et al., 2018;Miyake et al., 2016). Although hypertelorism, developmental delay, and intellectual disability are found in at least two of these four patients, our phenotypic comparison reveals that the patients with mutated AT hooks still lack representative or consistent features. There are three possible explanations for these observations: (i) the number of identified variants is small. (ii) the identified patients in this group are too small to show all the malformations. (iii) the impact of AT-hook variants may be relatively milder than that of the other two groups of variants studied.  . Wang et al. found that PHD fingers are capable of recognition of different histone marks, DNA binding, mediation of protein-protein interactions, and E3 ubiquitin ligase (Wang et al., 2012). Generally, the patients (including six reported patients and our patient 2) with mutated intermediate region between CXXC and PHD domain have more severe symptoms than those carrying variants in AT-hooks (Baer et al., 2018;Li et al., 2018;Strom et al., 2014). Among these seven patients, some symptoms (such as developmental delay, intellectual disability, craniofacial features of prominent forehead, hypertelorism, deformed palpebral fissures, malformed nasal bridge, and thin upper lip) are universally noticed, and moreover, some symptoms (such as thick eyebrow, long eyelashes, and hypertrichosis) are found to be in high frequency. Of note, one patient with p.Ser1299Profs*2 was found to have epilepsy, which is not frequently observed in the WSS patients (Chan et al., 2019). Accordingly, we speculate that the intermediate region between CXXC and PHD domain affect normal functions of the two domains during the development of multiple systems.
The majority of KMT2A variants associated with WSS have been truncating variants (Jones et al., 2012;Min Ko et al., 2017). However, missense variants are known to explain a significant fraction of the WSS patients observed. In our patient 3, we identified a novel likely pathogenic variant  (Baer et al., 2018;Lebrun et al., 2018;Min Ko et al., 2017;Stellacci et al., 2016). Among them, c.3460C>T (p.Arg1154Trp) was found to lead to significantly increased gene expression level, but not change the protein expression level (Lebrun et al., 2018), underlying the importance of CXXC domain. In accordance with the key regulation role of CXXC domain and severe neuro-phenotypes associated with the variants in this domain (Lebrun et al., 2018;Li et al., 2018;Min Ko et al., 2017), the WSS patients (seven reported patients and our patient 3) with mutated CXXC domain have more severe and profound anomalies than those with mutated AT hooks or intermediate region between CXXC domain and PHD domain (Table 1). Microcephaly (5/8 patients) as well as small hands and feet (6/8 patients) are prevalent in patients with mutated CXXC domain. In terms of neurodevelopment, language delay is presented in 7/7 patients, epilepsy detected in 3/6 patients, aggressive behavior exhibited in 3/7 patients and high severity level of intellectual disability found in 8/8 patients.
In summary, our study would aid in molecular diagnosis of WSS and expand our knowledge about genotypephenotype correlation of this disorder. WSS heterogeneities in both genotype and phenotype are evident in our patients herein described and in previous reported patients. Given that KMT2A variants is associated with multi-systematic anomalies, especially in heart, lung, brain, and immune systems, we recommend that multiple tests of spine MRI and CT, immunological examination, growth hormone determination, eye examination, ENT examination, cardiac ultrasound, abdominal ultrasound, EEG and cranial MRI examination, and genetic testing are helpful for assisting clinical diagnosis and management of the WSS patients.

ACKNOWLEDGMENT
The authors are grateful to all the patients and their families for their contributions to the study.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS
XH and LY conceived the study; SL, BB, YH, and XH clinically analyzed the patients; WZ and PZ conducted genetic data interpretation; WZ and XH performed literature review and prepared the manuscript; WZ, RZ, WC, and XH revised the manuscript. All authors reviewed and approved the manuscript.

ETHIC STATEMENT
This study was approved by the hospital's ethics committee.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are included within the article.