Wolf–Hirschhorn syndrome (WHS; OMIM 194190) is a rare genetic syndrome caused by terminal chromosome 4p deletions and is also known as 4p-syndrome. WHS is characterized by typical craniofacial features including a “Greek warrior helmet” appearance of the nose (wide flattened nasal bridge continuing to the forehead), microcephaly, widely spaced eyes, a distinct mouth, a short philtrum, micrognathia, downturned corners of the mouth, and poorly formed ears with pits and tags. Growth restriction, postnatal growth deficiency, hypotonia with muscle underdevelopment, and developmental delay/intellectual disability of variable degrees are observed in all affected patients. Other findings include heart and skeletal defects, seizures, abnormal tooth development, and hearing loss (1,2).

WHS is caused by a deletion in the 4p16.3 region, with deletions shorter than 3.5 Mb associated with a milder phenotype without major malformations, and two WHS critical regions—WHSCR−1 and WHSCR−2—overlap the WHSC1 gene (3–5). The WHS candidate 1 (WHSC1) gene, also known as NSD2 (OMIM 602952), encodes nuclear receptor-binding set domain protein 2 and play a important role in normal development (6). Recently, 36 novel or de novo variants in NSD2 variants were identified in 36 patients with overlapping yet atypical features of WHS, thus defining a novel WHS-like disorder, namely Rauch-Steindl syndrome (RSS,OMIM 619695) (Supplementary Table S1) (7–12). Patients with RSS exhibit a wide range of mild phenotypic features, with core manifestations of microcephaly, intrauterine growth restriction, facial dysmorphisms, autism, intellectual disability, and low birth weight, feeding difficulties, failure to thrive, short stature, speech delay and muscular hypotonia.In this study, In this study, we identified a novel de novo NSD2 gene variant [c.2721delT(p.Asn907Lysfs*5)] in a Chinese girl diagnosed with Rauch–Steindl syndrome. In addition, our study further expands the phenotypic spectrum of WHS and the mutation spectrum of NSD2.

NSD2 (WHSC1) is∼90 kb long and located on human chromosome 4p16.3, and encods the nuclear SET domain-containing transcriptional regulatory protein, which contains four development-related domains: a PWWP domain, an HMG box, a SET domain, and a PHD-type zinc finger. NSD2 is a SET domain histone methyltransferase responsible for the methylation of H3K36, is expressed widely across many tissue types, and participates in a variety of biological processes, including early development, cytokine signaling, the DNA damage response, and class switch recombination. Haploinsufficiency of NSD2 (pLi = 1.00) is thought to be to be an important part of the mutational mechanism of WHS. Heterozygous knockout-NSD2 mouse models show growth restriction, craniofacial malformation, and midline fusion defects (15, 16). Zanoni et al. demonstrated that loss-of-function and missense variants in NSD2 lead to reduced methylation activity and are associated with a distinct developmental phenotype (11). These findings suggest that NSD2-deficiency is responsible for multisystem abnormalities in patients with Rauch–Steindl syndrome. Recent reports have confirmed that patients with novel loss-of-function or missense variants in NSD2 exhibit a variety of abnormalities including facial dysmorphism, microcephaly, intrauterine and postnatal growth restriction, craniofacial malformations, failure to thrive, intellectual disability, speech delay, behavioral/psychological issues, and skeletal and limb abnormalities (7–12). In the present study, the de novo variant c.2721delT is located in the PWWP2 domain in NSD2 and is predicted to create a frameshift starting at codon 907 leading to a stop codon 5 positions downstream (p.Asn907Lysfs*5). This variant may result in the absence of protein production with a significant decrease in mRNA level due to nonsense-mediated decay degradation. The present patient showed common phenotypes associated with Rauch–Steindl syndrome, including facial dysmorphism, microcephaly, a degree of intellectual disability, growth restriction, severe malnutrition, short stature, small hands and feet, muscular hypotonia, mild clinodactyly of right hand, and loose skin on the hands and feet, and these phenotypes are thought to be due to the loss-of-loss-of-function variant detected in NSD2.

To date, 37 NSD2 variants have been identified, including ten missense variants (eight de novo, two inherited), fifteen frameshift variants (nine de novo, two inherited, four undetermined), ten nonsense variants (nine de novo, two undetermined), and two splice site variants (two de novo) (Supplementary Table S1). These variants are distributed across the entire gene, and 87.5% (28/32) are de novo (Supplementary Table S1, Figure 1). Of the 37 NSD2 variants, 11 (including our variant) located on the PWWP domain, including two missense variants (two de novo), four frameshift variants (three de novo, one undetermined), and four nonsense variants (three de novo, one undetermined). This is not significant compared to the variants in the entire gene. In addition, Rauch-Steindl syndrome is a highly heterogeneous disease caused by variants in the NSD2 gene. The phenotypes of the Rauch-Steindl syndrome are complex but several common characteristics are observed in most patients (11). Zanoni et al. have been reported that the core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS (11). Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. The results showed that the relationship between genotype and phenotype seemed to be related to the mutation type of the mutant, but not to the position of the mutant in the protein. Limited by the currently reported cases and variants, these results should be seen as provisional. Future studies with the report of increasing numbers of patients will therefore be necessary to to refine the phenotype and clarify genotypic effects and other phenotypic determinants.

Typical WHS has been described as a disorder with special craniofacial features, growth restriction, intellectual disability, seizures and skeletal abnormalities resulting from heterozygous deletions in the 4p16.3 region (1). Compared with typical WHS patients, an atypical mild manifestation was observed in our patient. In addition to our patient, other reported patients showed less marked craniofacial features of WHS. 60%–70% of typical WHS patients have severe skeletal anomalies including kyphosis/scoliosis with malformed vertebral bodies, accessory or fused ribs, clubfeet, and split hands. Only 39% of patients with a NSD2 variant have skeletal abnormalities, which is less severe than in typical WHS patients (9–11). Mild clinodactyly in the right hand of the present patient was observed. This indicates that the NSD2 gene may not be the main cause of WHS craniofacial features and skeletal anomalies, and it may have a cumulative effect on severe intellectual disability, typical craniofacial features, and skeletal anomalies of WHS with other genes located in the 4p16.3 region. Seizures are the most common problem in children with WHS (90%–100%) and are the greatest problems in the clinical management of WHS (17). As in previously reported patients with NSD2 variants, our patient also lacked seizures or seizure-like episodes, further indicating that seizures are not associated with the NSD2 gene and that other genes located within the 4p16.3 region such as LETM1 may be responsible for seizures (18). In addition, the severity of the intellectual disability in patients with NSD2 patients was milder compared with patients carrying the most common 4p deletions (which are between 5 and 18 Mb in length). Furthermore, the majority of individuals with NSD2 variants lack many of the most common manifestations of WHS, such as orofacial clefts and defects and genital, cardiac, and renal malformations. Therefore, NSD2-deficiency may be necessary for WHS, but it is not sufficient to cause WHS. In addition, genetic diagnosis is the best method for detecting the cause of the syndrome in these patients.

In conclusion, we found a de novo heterozygous frameshift pathogenic variant, c.2721delT(p.Asn907Lysfs*5), in the NSD2 gene in a Chinese girl with Rauch–Steindl syndrome using WES. Our findings support that this loss-of-function variant in NSD2 is a cause of a syndrome consisting of intellectual disability and developmental delay. Detailed clinical features and molecular diagnosis will further help our understanding of the phenotype-genotype correlations of NSD2 pathogenic variants and related disorders including WHS. In addition, molecular genetic testing of NSD2 is a useful tool for clinical diagnosis and genetic counseling of these patients. The novel pathogenic variant uncovered in this study expands the mutation spectrum of NSD2.