Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

Abstract

Purpose: Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf-Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood.

Methods: We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro.

Results: 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. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2's folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants.

Conclusion: NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch-Steindl syndrome after the delineators of this phenotype.

Fig. 1. Localization of the NSD2 variants.

The diagram shows the structure of the NSD2 gene (above) and protein (below, isoform 1, encoded by the NM_133330.2 transcript) together with the variants discussed in this study as well as variants that have previously been reported in large sequencing studies. The variants carried by the 18 additional individuals described in this study are in bold. Observed germline variants absent in the HGMD database are underlined. † Shared by more than one patient; # variant of uncertain significance (VUS)/likely benign. The observed pathogenic missense variants we found map to three distinct domains of NSD2: a PHD zinc-finger domain (residues 831–875), a PWWP domain (residues 880–942), and the catalytic methyltransferase domain (residues 1011–1203; composed of three subdomains, namely an AWS, a SET, and a post-SET domain). The color coding of the variants and protein domains is reported in the legend. AWS Associated With SET domain (IPR006560), GOF gain of function, HMG high mobility group box domain (IPR009071), LOF loss of function, MTD catalytic methyltransferase domain, composed by the AWS, SET and a post-SET domain, PHD zinc-finger domain, Plant-HomeoDomain type (IPR001965), PWWP proline–tryptophan–tryptophan–proline domain (IPR000313), SET Su(Var)3-9, enhancer-of-zeste, trithorax domain (IPR001214). Domains were annotated according to the Uniprot databank (see Web Resources).

Fig. 2. Structural and functional effect of two synthetic and four naturally occurring pathogenic NSD2 missense variants.

(a) Wild-type (WT) Cys869 is one of four cysteines that tetrahedrally coordinate a zinc ion (Cys846, Cys849, Cys869, Cys872 are shown in stick presentation; the Zn²⁺ is depicted as a brown ball). (b) The variant Tyr869 adopts a different sidechain orientation resulting in a loss of the zinc ion (marked by a red arrow). (c) WT Pro895 is located in spatial proximity of Trp885 (cyan). (d) The longer Leu895 sidechain present in the variant results in steric clashes with Trp885 (marked by a red dotted circle). (e) WT Ser1137 is located in spatial proximity of Leu1163 (cyan). (f) The bulkier Phe1173 sidechain present in the variant results in steric clashes with Leu1163 (marked by a red dotted circle). (g) WT Glu1091 forms a salt bridge to Arg1160 (green arrow), which is disrupted in the (h) Lys1091 variant and steric clashes are formed instead (red arrow). (i) WT Tyr1092 forms tight van der Waals interactions with Leu1120 (cyan), which are lost in the synthetic (j) Ala1092 variant (the site of altered interactions is denoted by a red arrow). (k) WT Tyr1179 forms stabilizing interactions to S-adenosylmethionine (SAM, cyan). (l) The shorter sidechain in the synthetic Ala1179 variant results in a loss of interactions with the SAM cofactor (red arrow), which is expected to result in a drastic loss of enzymatic activity. (m) Western analysis with the indicated antibodies of whole-cell extracts (WCEs) from 293 T cells overexpressing vector control, full-length WT NSD2, or NSD2 mutants as indicated. Histone H3 is shown as a loading control. (n) Quantification of western blot data in (m). (o) In vitro methylation assay with recombinant WT NSD2 or mutant NSD2 derivatives as indicated on recombinant nucleosomes (rNuc) as substrates. Top panel, ³H-SAM is the methyl donor and methylation is visualized by autoradiography and indicated as ³H-H3. Bottom panel, Coomassie stain of proteins in the reaction. (p) Quantification of all detectable bands in the autoradiography in (o). (q) Western analysis with the indicated antibodies of in vitro methylation assay with nonradiolabeled SAM. (r) Quantification of all detectable bands in the western blot data in (q). (s) Western analysis with the indicated antibodies of WCEs from WT or NSD2 deficient HT1080 cells complemented with CRISPR-resistant NSD2 (WT or mutants), or control as indicated. Histone H3 and tubulin are shown as loading controls. (t) Quantification of western blots in (s). The data in (n, p, r, t) are represented as mean ± SD of two independent experiments. *p < 0.05 based on a one-way analysis of variance (ANOVA) followed by two-tailed Dunnett’s test.

Fig. 3. Phenotypic features and growth parameters of the individuals carrying NSD2 pathogenic variants and comparison with Wolf–Hirschhorn syndrome (WHS).

The diagram in (a) summarizes the phenotypic features of the individuals described in this study (18 additional patients together with 10 previously reported individuals) based on the type of NSD2 variant carried. In (b) all individuals are compared with WHS patients, subdivided according to the size of the 4p deletion carried, as reported in Zollino et al. Data in (a) and (b) are expressed as percentages. Growth parameters at birth (c; included the parameters at termination of pregnancy for individual 16-I) and at last visit (d) are reported. The average measures for all combined individuals standard deviation score (SDS [SD]) were length (L): -2.3 (1.5); weight (W): -2.0(1.0); occipitofrontal circumference (OFC): -1.1(1.7); gestational weeks (GW): 38.5(3.8) at birth and height (H): -1.7(1.3); W: -1.7(1.5); OFC: -2.4(1.8); body mass index (BMI): -1(1.5) at last visit. (e) Comparison of the growth parameters at last visit for patients carrying missense variants (black) compared with patients carrying other variants as well as deletions encompassing NSD2 (red). *p value < 0.05 as tested by two-tailed Student’s t-test. The data in (c–e) are expressed as SDS based on the respective growth charts. Lines and whiskers represent mean ± SD. (f) Linear correlation analysis between age and BMI and last visit. ASD DD/ID developmental delay/intellectual disability, IUGR intrauterine growth restriction, SC spinal cord, SGA small for gestational age.

Fig. 4. Clinical data.

(a) Facial features of the affected individuals reported in this study. The age in years (y) and months (m) is reported beside each patient ID. (b) Combined facial gestalt obtained by combining frontal photos using the Face2Gene online research tool (see Materials and Methods and Web Resources). Photos from 12 different patients, including those depicted in (a), were used for this purpose. (c) Family tree of family number 6 in this study. M+/−/? = presence (+), absence (−), or unknown status (?) for the p.Asp1158Glyfs*11 variant in each NSD2 allele. Clinically affected individuals are shown as full symbols.