Genomics studies of neurodevelopmental disorders (NDD), in particular autism spectrum disorder (ASD), have uncovered rare but recurrent pathogenic  variants in a growing list of causal genes [1,2,3]. The clinical application of genomic copy number variants (CNVs) exome sequencing (ES), and whole genome sequencing (WGS) are routinely offered to patients with ASD and other NDD [4]. Approximately 15% of patients with ASD are found to have an identifiable genetic cause involving about 200 genes [1,2,3,4]. Notably, pathogenic variants in a significant number of genes encoding proteins in epigenetic machinery have been found in patients with ASD and NDD [5,6,7].

After the evaluation of the patients in clinic, we have identified protein truncating variants (PTVs) of MSL2 (male-specific lethal 2 in Drosophila) ES in three subjects with ASD and developmental disorders (DD). MSL2 encodes a protein that is a subunit of a protein complex for dosage compensation for sex chromosome specific genes in Drosophila [8]. MSL2 is implicated in histone modifications through ubiquitination and acetylation [9, 10]. The discovery of predicted loss-of-function variants in MSL2 supports the pathogenicity of these PTVs for NDD and ASD and an as yet undefined important function of MSL2 in brain development.

Clinical presentations and genetic findings

Three unrelated cases were evaluated in clinics. Case 1 had clinical trio ES. Patients 2 and 3 had proband-only ES. Clinical information was provided by the referring physicians or extracted from the clinical notes.

Patient 1

This patient is a 6-year-old girl with ASD, DD, and other behavioral problems including anxiety and self-injurious behavior (Unpublished abstract, Dept. of Pediatrics, Duke University). She was a product of a term pregnancy and normal delivery. Her newborn course was complicated by central hypotonia and feeding difficulties. Family history was significant for learning difficulties in the father and maternal half-brother. Her current weight was 18.9 kg (47th percentile), height was 118.2 cm (17th percentile) and head circumference was 49.6 cm (96th percentile). Overall, her motor development was delayed. Mild facial dysmorphism was noted with the features of down-slanting palpebral fissures, webbed neck, flat midface, low-set ears, micrognathia, down-turned mouth, and webbed neck. The diagnosis of ASD was confirmed with ADOS-2. Her standard score on WPPSI-IV was 75 (5th percentile). The score for Visual Perception was 45 (2nd percentile), for the Motor Coordination was 63 (1st percentile) and Visual Motor Integration was 77 (6th percentile). The score on PPVT-IV test was 80 (9th percentile). The performance on Visuomotor Precision Time was average (score = 3), but on the Visuomotor Precision Combined, it was extremely poor (score = 3). No history of seizure. MRI of brain was unremarkable. The comparison of clinical features for 3 patients was summarized in Table 1.

Table 1 Summary of clinical features associated with MSL2 PTVs.

Patient had negative FMR1 and chromosome microarray (CMA). Trio ES identified a de novo indel of c.796-797delCT in the MSL2 gene (NM_018133.3) (Fig. 1A, B). The open reading frame (ORF) analysis predicted that mutation results in a frameshift and truncated protein (p.Leu266Valfs*5). The truncated protein, if stable, would miss the C-terminal CXC domain of E3 ubiquitin-protein ligase of MSL2 protein (Fig. 1C).

Fig. 1: Schematic illustrating MSL2 gene, mRNA, and protien structure and as well as annotatoins of pathogeneic variants.
figure 1

A MSL2 gene structure and postions of pathogentic variants. B mRNA of MSL2 (Noted: NM_018133.3 was used to annotate the variant of patient #1 and NM_018133.4 was used for the patient #2 and #3). C MSL2 protein structure and pathogenic variants.

Patient 2

The patient is an 11-year-old boy referred for the evaluation of global developmental delay, intractable seizure, abnormal brain MRI, and behavioral abnormalities. The pregnancy was complicated by an abnormal prenatal ultrasound showing brain abnormalities. He was born at full term via repeat C-section. Growth parameters were normal. He walked around 15 months of age and developed words between 2 and 3 years of age. He has self-injurious and aggressive behavior. Seizure started at 18 months of age and was not fully controlled with the seizure medications. MRI of brain showed cystic changes in the right subcortical and periventricular white matter. He also has left hemispheric atrophy with ex vacuo dilatation of the left lateral ventricle, and cerebellar atrophy. EEG was abnormal with dysrhythmia and bitemporal discharges. Mild dysmorphisms include hypertelorism, epicanthal folds, slightly down-slanting palpebral fissures, flat midface, mild low-set ears, wide and smooth philtrum, and micrognathia (Fig. 2). The pectus excavatum were also noted (Table 1).

Fig. 2
figure 2

Front and side facial profile of patient 2 showing slightly down-slanting palpebral fissures, flat midface, mild low-set ears, wide and smooth philtrum, and micrognathia.

The patient had negative CMA, FMR1, and biochemical tests. ES identified a 4 bp deletion of c.1047_1050delTGAG in the MSL2 gene (NM_018133.4) (Fig. 1A, B). The ORF analysis predicted that variant results in a truncated protein of p.Ser349Argfs*23 (Fig. 1C). The truncated protein, if stable, is predicted to miss the CXC domain of E3 ubiquitin-protein ligase (Fig. 1C). Parental test of mother was negative but father was not available for the test.

Patient 3

This patient is a 16-year-old female with central hypotonia, exercise intolerance, and difficulty swallowing. She was born at full term via cesarean section with no perinatal complications. She did not have facial dysmorphism. She had feeding issues and required soft or pureed diet. She walked at age 16 months and motor development was delayed. Central hypotonia was first noted at 18 months of age and she has since regressed her motor milestones. Growth was normal. She had normal language development. No significant cognitive impairment and abnormal behaviors were reported. She was ambulatory with significant weakness at teenage that required wheelchair assistance. She had hyper-extensibility of joints. Her muscle CPK was normal. Electromyography, nerve conduction study, ECHO, and EKG were negative. Her polysomnogram was normal. She had mild thoracic scoliosis ( < 10 degrees). There was a family history of similar symptoms in the mother and maternal grandmother. Muscle biopsy in mother was reported as negative.

ES revealed a heterozygous novel nonsense variant in exon 1 (c.67 G > T, p.Gly23*) of the MSL2 gene (NM_018133.4) (Fig. 1A, B). The open reading frame (ORF) analysis predicted that mutant protein miss all known functional domains for MSL2 protein (Fig. 1C). Parental test for the variant was not completed due to family relocation.

The summary of PTVs of MSL2 from other genomic studies of ASD, NDD, and DD

We found a total of 12 de novo PTVs of MSL2 from 3 large scale of genomics studies (Table 2). In the study by Kaplanis et al. [11], 45,221 coding and splicing de novo mutations (DNMs) in 31,058 individuals were pooled from 3 cohorts: GeneDx (GDX), the Deciphering Developmental Disorders (DDD) study and Radboud University Medical Center (RUMC). Nine de novo PTVs and 3 missense variants of MSL2 were described from these cohorts with DD. In study by Zhang et al. [12], a targeted sequencing of 547 genes was performed for 1102 subjects with NDD. Two de novo PTVs of MSL2 were identified. In the study by Iossifov et al. [13], ES of 2508 subjects with ASD was performed. One PTV of MSL2 was identified. Unfortunately, the specific clinical features other than DD/NDD/ASD were not described in these studies. There are 3 PTVs (2 de novo) deposited in ClinVar (Table 2). The indication for genetic testing is ASD for 2 cases and global developmental delay for other case. Collectively, the report of these MSL2 PTVs provide additional evidence supporting the causal role of MSL2 in ASD/NDD.

Table 2 Summary of MSL2 PTVs from other genomics studies of ASD, NDD, and DD and ClinVar.


We presented, for the first time, detailed clinical presentations of 3 patients with PTVs of MSL2. We also identified three PTVs in ClinVar that are classified as variant of uncertain significance (VUS). The indication of genetic testing for two cases is ASD and other is DD. Together with 12 de novo PTVs of MSL2 curated from 3 large cohorts of clinical genetic testing or research studies, our finding provides strong support for a pathogenic role PTVs of MSL2 in NDD and ASD. The detail clinical features associated with PTVs of MSL2 are not available in other cases. Because of the report of dysmorphic facial features in our cohort, pathogenic variant in MSL2 may represent a syndromic NDD. Similar to many recent discovered genes implicated in NDD and ASD, the clinical features associated with PTVs of MSL2 are variable. For example, no apparent cognitive impairment was observed patient 3 with the caveat that the patient is lost for follow up. This is somewhat unexpected because nonsense variant at very beginning of coding exon in patient 3 is predicted to result in complete loss of function of MSL2 protein. Further neurodevelopmental evaluation or nature history are warranted. In a recent report, the protein truncating variants of MSL2 are characterized as nonsense-mediated decay escaping variants (ASHG abstract#335 2022). These findings suggest that PTVs of MSL2 do not necessarily result in a loss of function mechanism as predicted. The possibility that the truncating variant at C-terminus result in a gain of function mechanism could be considered because of the presence of a stable and truncated MSL2 protein with zinc finger domain in the N-terminus. The similar mechanism has been described for PPM1D truncating mutations associated NDD [14]. The genotype phenotype correlation in cases with MSL2 PTVs may be more complex and required more functional studies.

MSL2 was first identified in Drosophila, designated male-specific lethal 2. MSL2 is only expressed in male Drosophila and encodes a protein that functions as part of a multi-subunit dosage compensation complex on the X chromosome [8, 15]. The function of human MSL2 remains poorly characterized. In mammalian cells, MSL2 is a subunit of a protein complex including MSL1, MSL2, and MSL3 that implicates in chromatin modifications [16, 17]. MSL2 is implicated both in acetylation of histone H4 on lys16 (K16) [9] and in ubiquitination of histone H2B on K34 (H2Bk34) [10]. H2BK34 ubiquitination increased H2BK120 ubiquitination indirectly by facilitating chromatin association of both RNF20 and RNF40 [9, 18]. To our knowledge, this is the first example that two different types of histone modifications mediated by a single protein and implicated in NDD. Recent studies of MSL3, a close family gene of MSL2, have shed some light into the importance of the MSL complex in neurodevelopment. Pathogenic mutations of MSL3 have been reported in two cohorts with Basilicata-Akhtar syndrome [19, 20]. Majority of variants in MSL3 are de novo but rare inherited variants are also observed. The reduction of histone acetylation of H4K16 is seen in skin fibroblasts of these patients [19]. The clinical features associated with MSL2 and MSL3 pathogenic variants overlap. These include the developmental delay, intellectual disability, ASD, hypotonia, epicanthal folds, low set of ears, pectus excavatum and MRI brain finding of dilation of ventricle. Further functional studies of variants in MSL2 patients are necessary to investigate the perturbations induced by these variants in MSL2.