A novel nonsense variant in NSD1 gene in a female child with Sotos syndrome: A case report and literature review

Abstract Introduction Sotos syndrome (SS) is an overgrowth disease characterized by distinctive facial features, advanced bone age, macrocephaly, and developmental delay is associated with alterations in the NSD1 gene. Here, we report a case of a 4‐year‐old female child with SS caused by NSD1 gene nonsense mutation. Methods Whole‐exome sequencing (WES) was applied for probands and her parents. Sanger sequencing was used to confirm the mutation. We performed the literature review using PubMed and found 12 articles and 14 patients who presented with SS. Results The patient showed typical facial features of SS, hand deformities, and seizure. WES revealed de novo heterozygous variant: NSD1 (NM_022455.5), c.6095G > A, p.TRP2032*. We also reviewed the phenotype spectrum of 14 patients with SS, who exhibited a variety of clinical phenotypes, including developmental delay, seizures, scoliosis, hearing loss, cardiac and urinary system abnormalities, and so on. Discussion The lack of correlation between mutation sites or types and phenotypes was summarized by literature reviewing. The NSD1 protein contains 14 functional domains and this nonsense mutation was located in SET domain. Early appearance of the termination codon leads to protein truncation. Haploinsufficiency of the NSD1 gene causes the overgrowth disorders.

been reported that up to 90% of patients are involved in alterations in the nuclear receptor binding SET domain protein 1 (NSD1) gene.
The nonsense mutation c.6095G > A (p.TRP2032*) in NSD1 gene causing SS has not been reported before.It is concluded that this intragenic truncating variant was pathogenic and changed the protein structure.

Trio-based whole-exome sequencing
Trio-whole-exome sequencing (WES) was performed for probands and her parents.Peripheral blood was extracted from all probands and their parents into EDTA anticoagulant tubes.The Agilent SureSelect was used to capture exons (Exome V6).After establishing the libraries, the data were sequenced on Illumina.Human Gene Mutations database (HGMD), Ingenuity online software system, and ClinVar database were employed to annotate related diseases.The candidate variations were verified by Sanger sequencing.

Literature review
The literature review was conducted by PubMed database using the terms ("Sotos syndrome" OR "Sotos' Syndrome" OR "Soto Syndrome" OR "Soto's Syndrome" OR "Cerebral Gigantism") AND "NSD1".Publications from 1993 to 2023, including NSD1 gene point mutations, ages from 0-18-year old and detailed description of patient characteristics were selected.

Case details
The 3-year-and-11-month-old female child is the second child of nonconsanguineous healthy parents and she has a healthy 10-year-old elder brother.She was born by full-term cesarean section and her birth weight was 3300 g with a head circumference greater than the normal range.She had hand deformity manifested as flexing hands, and the fingers tilted outward could not be extended.
At 5 months, she was not able to look up or turn over.After rehabilitation training, she could walk with something and said simple words such as "Mom" and "dad."After that, she stopped rehabilitation training.At the age of 3, she could walk and run alone, however, still could only speak some simple words.Currently, her height was in the 97th percentile of normal peers, and her weight was greater than the 97th percentile of normal children with the same age.She had the regression of language development and was unable to communicate routinely.
She had a typical facial appearance, with protruding forehead, 57 cm of head circumference, oblique cleft eyes, widely spaced between eyes, long tip of mandible, and degeneration of hair on both temporal areas.
Except for the thumb, the other four fingers of both hands were in the excessive outer booth, and the palms of both hands had no palm print.
The first seizure occurred when the child was 3-and-a-half-year old, and then repeated attacks, manifested as cyan lips, dyspnea, stiffness, accompanied by fever, which relieved itself after 2-3 min.
De novo heterozygous variant was found by WES in trios: NSD1 (NM_022455.5),c.6095G > A, p.TRP2032*.This mutation was pathogenic in accordance with the American College of Medical Genetics and Genomics criteria (ACMG) guidelines.
EEG showed abnormal discharges in occipital area (Figure 1).MRI displayed that the ventricle was slightly larger (Figure 2).According to the clinical manifestations of the patient, oxcarbazepine was given to control seizures.Rehabilitation training and regular follow-up were required.The female child was diagnosed with SS on the basis of clinical manifestations, imaging, and genetic examination.

Literature review
We retrospected 12 articles and 14 children with SS caused by alterations of NSD1 gene were included.The median age of the patients was 2-and-a-half-year old (range 8-month to 10-year old).The characteristics of 14 cases with SS were summarized through reviewing the literature shown in Table 1.The incidence of SS in male is higher than female (10:4).Most cases are sporadic, but some familial autosomal dominant cases have also been described (2/14) (Leventopoulos et al., 2009).Loss function of NSD1 gene is due to microdeletion and mutations (missense variants, nonsense variants, splice variants, small insertions, and rearrangements) (Mencarelli et al., 2018), mainly leading to macrocephaly and overgrowth (12/14).Almost patients presented with development milestones delay in speech, dyskinesia, and different degrees of learning impairment (14/14) (Han et al., 2017;Piccione et al., 2011).Many cases were reported other clinical features, such as scoliosis, renal malformations, cardiac anomalies, syndactyly, neonatal hypotonia, joint hyperlaxity, reproductive system diseases, seizures, myopia, and conductive hearing loss (Foster et al., 2019;Sohn et al., 2013;Tatton-Brown et al., 2005).Neuroimaging examinations commonly reported the ventricular enlargement, midline structure anomalies, and hypoplasia of the corpus callosum (Piccione et al., 2011).There is no correlation between genotype and phenotype, because cases with the same mutation sites usually have different clinical manifestations (Baujat & Cormier-Daire, 2007).

DISCUSSION
In our case, the female patient had no family history, and the variant was de novo.This patient had typical facial abnormalities including   reports (Grand et al., 2019;Sohn et al., 2013).In addition to the typical facial features of SS, she also showed hand deformities.Her height and weight were in the 97th percentile of normal peers, which is the representative feature of SSs.Growth velocity is higher than peers in their childhood, as adults, their height usually falls within the normal range (Özcabi et al., 2020).The girl did not perform organs damage like congenital heart disease, kidney repair.But she had developmental delay and febrile seizure.SS is an overgrowth disease, but many patients also suffer from epilepsy.The seizures are usually self-limited.Staring spells were the most frequent seizure type, followed by febrile seizures and afebrile bilateral tonic-clonic seizures (Fortin et al., 2021).SS also can be complicated by mental and psychological problems, such as anxiety, depression, attention-deficit hyperactivity disorder, and autism spectrum disorder (Özcabi et al., 2020).These symptoms were absence in our case.
The main function of NSD1 gene is transcriptional activation and repression through chromatin modification.It is a histone methyltransferase that primarily dimethylates Lys-36 of histone H3 (H3K36me2), trimethylates Lys-20 of histone H4 (H4K20me3) as well (Watanabe et al., 2020).The methylation of H3K36 marks the expressed genes and is related to the inhibition of the initiation of intragenic transcription in the expressed genes (Lee & Shilatifard, 2007;Pasillas et al., 2011).H4K20 methylation participated in serious activities, including mitosis, gene activation and repression, chromatin condensation, and DNA-damage checkpoint signaling (Rayasam et al., 2003).Another function of NSD1 gene is acting as a co-inhibitor of growth promoting genes, which will have a positive or negative impact on the transcription of nuclear receptors (e.g., estrogens, retinoic acid, and thymoid hormone receptors) depending on the cellular environment (Ha et al., 2016).In many cases, haploinsufficiency of NSD1 like nonsense or missense mutations and deletions was suggested to be associated with overgrowth in stature and face.Thus, the main cause of SS is the haploinsufficiency of NSD1 gene in the distal long arm of chromosome 5 (5q35.2-q35.3)(Kurotaki et al., 2002).But in some cases, the 5q35 microduplication was confirmed and the patients manifest "reversed SS" as short stature and microcephaly without facial features (Franco et al., 2010;Reis et al., 2017).
The NSD1 contains multiple functional domains, including two nuclear receptor interaction domains (NID − L and NID + L), Pro-Tryptophan-Proline (PWWP) I and II, six plant homeo domains (PHD), SET-associated Cys-rich (SAC), SET, post-SET, and a PHD fingerlike Cys-His rich domain (Berardi et al., 2016;Tatton-Brown & Rahman, 2004;Türkmen et al., 2003).The main functional domains have been shown in Figure 3. NID − L and NID+L are corepressors and coactivators, respectively.Two different nuclear receptor interaction domains permit NSD1 to negatively and positively regulate transcription (Tatton-Brown et al., 2005).SET is a highly conserved catalytic domain, which is the most important area of NSD1.Other domains, such as PWWP and PHD, are auxiliary (Qiao et al., 2011).It suggested that PWWP domains are involved in protein-protein interactions, they act as the recognition sites of other proteins on NSD1, thereby acti-vating methyltransferase activity (Watanabe et al., 2020).PHD finger domains have effects on chromatin mediated transcriptional regulation (Türkmen et al., 2003;Verma et al., 2021).SAC domain may play a part in chromosome binding (Türkmen et al., 2003).In this case report, the nonsense mutation attacks in SET domain which is the core during methylation.SET can catalyze the transfer of methyl groups to lysine residues at the tail of histones (Watanabe et al., 2020).Due to the early appearance of the termination codon, the truncated protein lost post SET loop and two PHD fingers.Post SET loop is essential for the methyltransferase activity of NSD with nucleosome as substrate.A post-SET extension is responsible for nucleosome binding (Allali-Hassani et al., 2014;Graham et al., 2016).Berardi et al. (2016) indicated that PHD V combining with PHD VI can interact with the C2HR region of Nizp1, a transcription inhibitor.Nonsense mutations are able to make the truncated protein to be unstable and swift degradation after synthesis, that is, nonsense mediated degradation (NMD).We have mentioned above that haploinsufficiency of NSD1 is the major cause of SS.The nonsense mutation we reported was likely to have triggered the NMD mechanism, leading to rapid degradation of the faulty RNA, avoiding truncated proteins in large quantities (Chang et al., 2007;Holbrook et al., 2004;Malan et al., 2010).We deduced that these factors mentioned above account for the pathogenicity of the c.6095G > A (p.Trp2032*) variant.

CONCLUSION
We report a female child with SS carrying a de novo nonsense mutation in NSD1 gene.The phenotypes of SS and the function of NSD1 protein and each domain were summarized by literature reviewing.

F
Typical facial appearance of the case.(A) Prominent forehead, high hairline, and frontotemporal sparse hairs.(B) For both hands, the fingers were in the excessive outer booth, and the palms had no palm print.a prominence of the forehead, frontotemporal hair sparsity, downslanting palpebral fissures, flushing, and a pointed chin similar to other

F
Schematic representation of the functional domains in NSD1 protein.Distinct colored boxes represent different domains.The 23 boxes present with exons and introns are shown as lines with the exon numbers underneath and the domain names above.The location of the novel nonsense mutation is pointed out.
The heterozygous mutation c.6095G > A (p.Trp2032*) occurred on exon 20 of NSD1 gene and was located in the SET domain.It has never been described before and cannot be searched in various databases (e.g., Exome Variant Server, clinivar, HGMD, and gnomAD).According to the ACMG, the c.6095G > A (p.Trp2032*) variant can be classified as likely pathogenic.The nonsense variant led to the codon TGG encoding tryptophan at position 2032 changed into the stop codon TGA, and the early appearance of the stop codon at amino acid 2032.As a result, large fragments of NSD1 protein were truncated.