Neurodevelopmental disorders (NDDs) comprise a heterogeneous group of disorders including intellectual disability (ID) and autism spectrum disorders (ASDs) 1 . Approximately 1:200 to 1:450 children are born with a de novo pathogenic gene variant causing a Mendelian NDD 2 . Interestingly, recent large-scale exome sequencing studies have shown that defects in histone methyltransferases (HMTs) are major contributors to ASD and ID 3, 4 .
One of the genes involved in epigenetic modification through histone methylation is SETD1A (also known as KMT2F), which encodes a histone methyltransferase that mediates trimethylation of histone H3 on lysine 4 (H3K4me3) at promoters of active genes 5 . SETD1A methyltransferase activity depends on interactions with other proteins in a highly conserved complex, called 'complex of proteins associated with Set1' (COMPASS). COMPASS was originally defined in yeast 6 , and subsequently shown to be conserved in flies and mammals [7] [8] [9] .
SETD1A appears to be important for multiple aspects of cell cycle regulation. For example, SETD1mediated H3K4me3 activates β-catenin expression, which is required for proliferation of neuronal progenitor cells 10 . Furthermore, SETD1A has been shown to promote cell cycle progression by activating the expression of micro RNAs that suppress the antiproliferative gene BTG2, which plays a role in the transition between the G1 and S phase of the cell cycle 11 . Finally, H3K4 methylation by SETD1A is crucial in maintaining genome stability, especially during DNA replication, by protecting newly-replicated DNA from degradation 12 .
Heterozygous Loss-of-Function (LoF) variants of SETD1A have been identified in several human individuals from large schizophrenia cohort studies and have been linked to disruption of speech development and early-onset epilepsy [13] [14] [15] . Previously, LoF variants were also identified in a limited number of children with developmental delay 14 , suggesting that SETD1A variants are a common biological underpinning of the different aforementioned phenotypes. To provide further insight into the somatic and behavioral profiles, we characterised the neurodevelopmental syndrome associated with SETD1A heterozygous LoF variants in this study. This is a prerequisite to develop future guidelines and management directed towards the vulnerability to schizophrenia or other psychiatric diseases. In addition, to further establish the role of SETD1A in underlying biological processes, we studied the effect of SETD1A variants on genome stability in patient cell lines, and investigated the role of the Drosophila Melanogaster orthologue, Set1, in postmitotic memory forming neurons of the fly brain.
We collected the molecular and clinical features of 15 unpublished individuals with de novo SETD1A variants by a collaboration facilitated by GeneMatcher 16 in which several clinical groups independently identified individuals with developmental delay/intellectual disability (DD/ID) and related phenotypes with rare variants in SETD1A during routine diagnostic genetic testing. Clinical analysis of these patients was performed during regular consultations focusing on medical history, physical examination, and observational analysis of behavioral features. In all patients, exome sequencing and variant filtering were performed, according to the routine procedures at each institute [17] [18] [19] [20] [21] [22] .
We compared these data to available information from previously published cases with pathogenic SETD1A variants associated with schizophrenia and NDDs 14, 23, 24 . Ten individuals reported in these previous studies originate from cohorts diagnosed with schizophrenia (UK10K-Finns and Swedish cohorts) and six individuals originate from cohorts of individuals seen with NDDs (Deciphering Developmental Disorders (DDD) project, Northern Finland Intellectual Disability (NFID) study and Northern Finland Birth Cohorts (NFBC)). Clinical information of the previously reported individuals was only scarcely provided.
To further assess the impact of these variants on cellular SETD1A functions, we first isolated and generated lymphoblastoid cell lines (LCL) from 3 patients (6, 13 and 15) , and from two unrelated controls.
For further details on the methods used for cell culture, immunoblotting, antibodies and RNA isolation see Supplementary Data 1 -Methods.
DNA fibre analysis was carried out as described previously 12 . Briefly, cells were pulse-labelled with CldU and IdU (Sigma-Aldrich) for 20 min each before a 5 h exposure to 4 mM HU, and at least 200 replication forks were analysed per experimental condition. Images were taken using a Nikon Eclipse Ni microscope equipped with a 60X oil lens, and were acquired and analysed using Elements v4.5 software (Nikon). The lengths of labelled tracts were measured using ImageJ 25 and arbitrary length values were converted into micrometers using the scale bars created by the microscope.
To study the role of Set1 in postmitotic memory forming neurons of the fly brain, flies were reared on a standard medium (cornmeal-sugar-yeast-agar) at 25C and 70% humidity with a 12 h: 12 h light/dark cycle. R14H06-Gal4 flies express Gal4 under the control of a mushroom body (MB) specific enhancer from the rutabaga gene 26, 27 . mCherry RNAi , Set1 RNAi1 , Set1 RNAi2 , and Set1 RNAi3 were all generated by the Transgenic RNAi Project at Harvard University Medical School 28 using a common donor strain, therefore these RNAi lines all share a common genetic background. mCherry RNAi was used as a control in these experiments because it shares a common genetic background with all of the tested RNAi lines, and represents any non-specific effects of a non-targeting RNAi. mCherry RNAi , Set1 RNAi1 , Set1 RNAi2 , and Set1 RNAi3 were crossed to R14H06-Gal4 for courtship conditioning and activity experiments, actin-Gal4 for lethality assays, and UAS-mCD8::GFP; R14H06-Gal4 for analysis of MB morphology. For more details on the acquisition of the used fly stocks, see Supplementary Data 1 -
Flies were trained and tested for short-term (STM) and long-term memory (LTM) 4 days after eclosion using the courtship conditioning assay as previously described [29] [30] [31] . For each fly pair a courtship index (CI) was calculated, which is the proportion of time spent courting over 10 minutes.
The learning Index (LI) represents the percentage of reduction in courtship behavior due to training and is used to directly compare memory ability between different genotypes. LI is a single value that is calculated from the CIs using the formula: LI= (mean-CInaïve -mean-CItrained)/ mean-CInaïve. For more details on the courtship conditioning procedures, see Supplementary Data 1 -Methods.
One-to five-day old flies were assayed for locomotor activity in 12 h: 12 h light/dark regimens after at least three days entrainment, for four full days using DAM5M monitors (Trikinetics, Waltham, MA). Locomotor data was collected in 1 minute bins, and a 5 minute period of inactivity was used to define sleep 32, 33 . Sleep parameters were analysed using pySolo and plotted 34 . Dead animals were excluded from analysis by a combination of automated filtering and visual inspection of locomotor traces.
Brains from one-to five-day old day old adult male flies expressing the membrane bound UAS-mCD8::GFP under the control of R14H06-Gal4 were dissected in 1x PBS. Dissected brains were fixed in 4% paraformaldehyde for 50 minutes and then washed in a PBS with 1% Triton X-100 for 5 minutes. Brains were then mounted using Vectashield and imaged with a Zeiss LSM 710 confocal microscope. Confocal projections were created using ImageJ software (v. 2.0.0) 25 .
An extensive overview of the clinical and molecular data of the patients included in this study is provided per individual in Supplementary Table 1 . For one variant paternal genotype data was unavailable, therefore paternal inheritance could not be fully excluded. All other variants of the SETD1A gene identified in our cohort had occurred de novo. The 15 SETD1A variants identified here include 5 nonsense, 6 frameshifts, 1 missense and 2 different splice site variants, of which one occurred in two unrelated individuals. These 14 different identified SETD1A variants were all located before the conserved SET domain, which is responsible for catalyzing H3K4 methylation ( Figure 1 ).
To gain further insight into the pathogenicity of the missense variant (Tyr1499Asp), we utilized several in silico prediction models. MetaDome is an online data platform that uses homologous domain relations and combines data from GnomAD with ClinVar to produce a 'tolerance landscape' of a gene 35, 36 . According to MetaDome the region in which the variant lies is intolerant (Supplementary Figure 1 ). In addition, several other in silico prediction tools indicated that this variant is pathogenic: CADD PHRED: 29.7 37 ; SIFT: Deleterious, score 0 38 ; Polyphen-2: Probably damaging, score 1,000 39 .
In each patient, the observed SETD1A variant was considered to be the most likely contributor to the phenotype. In general, SETD1A is depleted from truncating variants in healthy individuals (pLi=1). The Cellular Impact of SETD1A Variants Immunoblotting analyses revealed that cells from two patients, harboring the p.Gly535Alafs* and c.4582-2_4582delAG variants, exhibited decreased levels of SETD1A protein. The presence of the Tyr1499Asp variant had no effect on SETD1A protein levels ( Figure 2A ). Moreover, when these patient-derived cells were exposed to DNA damage in the form of high-dose hydroxyurea, they exhibited slightly elevated activation of the DNA damage response compared to WT counterparts, evidenced by increases in phosphorylation of CHK1 and RPA ( Figure 2B ).
More strikingly, SETD1A variants were also associated with severe levels of nascent DNA degradation upon DNA damage, to levels comparable with depletion of SETD1A 12 . This was not evident in control cells, but was apparent in all 3 patient-derived cell lines examined ( Figure 2C ). These data suggest that these variants, including the Tyr1499Asp variant within the catalytic SET domain, may all lead to loss of SETD1A function. Moreover, given the severity of this defect, heterozygous variants in SETD1A mimic phenotypes that were previously observed in cell lines treated with SETD1A siRNA.
Our cohort of 15 individuals with heterozygous SETD1A variants comprised 8 females and 7 males whose ages varied from 34 months to 23 years (Supplementary Figure 3) . The collection of clinical data of these individuals has allowed us to further define the clinical phenotype associated with SETD1A haploinsufficiency. A summary of the clinical data of the 15 patients included in this study is presented in Table 1 . Figure 
Because of the previously reported association between schizophrenia and variants in the SETD1A gene 14 , we specifically looked at psychiatric and behavioral abnormalities in our patient cohort.
Behavioral abnormalities were observed in 14/15 individuals. These consisted of aggressive behavior (5/14), anxiety (4/14), short attention span (4/14), autistic features (3/14), overfriendliness (3/14) and abnormal temper tantrums (2/14). In our cohort, 7 out of 12 patients suffered from sleep disturbances, including night terrors.
There were signs (though no formal diagnoses) of a psychotic disorder in two individuals in the cohort. First, case 15, a 23-year-old man with the recurrent splice acceptor change (c.4582-2_4582delAG), experienced periods with apparent negative symptoms associated with schizophrenia (somberness and social withdrawal), alternating with periods of euphoria. He was treated with aripiprazole, 10 mg daily. He also showed aggressive and addictive behavior from a young age and has been diagnosed with PDD-NOS (DSM-IV criteria), which is part of the autism spectrum according to current diagnostic criteria (DSM-V) 1 . Second, case 3, a 16-year-old female, showed symptoms that could be classified as visual hallucinations and imperative auditory hallucinations, as she was reported to "hear voices telling her to do things and see things that are not there". This seemed to be transient and has not reoccurred. Because she was assessed as doing well, she so far has received neither formal evaluation nor medication for these symptoms.
Seizures were present in 3/15 individuals, with some very severe forms. For example, case 2 had myoclonic-astatic epilepsy. Drug treatment was sufficient to control seizures in case 2 and 3, but for case 11 hemispherectomy was required for seizure control. Hypotonia was reported in 9/15 individuals, often manifesting during infancy and remained relatively mild. In 4/12 patients for which MRI has been performed, morphological abnormalities of the brain were reported including corpus callosum abnormalities, cerebral white matter abnormalities, T2 hyperintensities, and Chiari I malformation. Other reported neurological abnormalities are EEG abnormalities, encephalopathy, dysarthria, slight tremor, sensory processing disorder, and short-term memory deficit.
Musculoskeletal abnormalities mainly affect the extremities (12/15) and included pes planus (6/12 
To gain insight into the function of SETD1A in neurons, we investigated Drosophila Set1, which is an orthologue of human SETD1A and SETD1B. Both SETD1A and SETD1B are mutated in individuals with overlapping neurodevelopmental disorders 41 . The Set1 family of proteins are known to be important regulators of cell proliferation and differentiation 10-12, 42, 43 . However, phenotypes that are normally associated with altered cell proliferation, i.e. overgrowth and cancer, are not frequently observed in SETD1A or SETD1B patient cohorts. To gain insight into the role of Drosophila Set1 in other aspects of neurobiology, we investigated terminally differentiated postmitotic neurons of the Drosophila mushroom body (MB), the primary learning and memory center of the fly brain.
Since germline Set1 mutants are lethal 44 , we used the spatially restricted Gal4/UAS system 45 to induce targeted RNA interference (RNAi) of Set1. Postmitotic MB neurons were targeted using the R14H06-Gal4 driver line, which we have characterised previously 26, 27 . R14H06-Gal4 is highly specific for the MB in adult and larval fly brains 46 . It is also expressed in a few cells of the ventral nerve chord, which appear to be chordotonal sensory neurons involved in balance and locomotion. We obtained three different Set1 UAS-RNAi transgenes that target different parts of the Set1 mRNA. We tested the RNAi potency by crossing RNAi stocks to the ubiquitous actin-Gal4 driver. As Set1 is an essential gene this would be expected to induce lethality. While Set1 RNAi1 and Set1 RNAi2 did induce lethality, Set1 RNAi3 did not, suggesting that the Set1 RNAi3 line is less potent. As a control, we used an mCherry RNAi strain (mCherry RNAi ) that had the same genetic background as the Set1 RNAi strains.
We tested the effect of postmitotic Set1 knockdown on memory using the courtship conditioning paradigm, a classic memory assay based on naturally occurring courtship behaviors 29 showing a significant reduction in CI in response to sexual rejection, and no significant reduction for Set1 RNAi1 and Set1 RNAi2 . Set1 RNAi1 and Set1 RNAi2 showed a significantly reduced LI when compared to the control for both STM and LTM, while Set1 RNAi3 did not ( Figure 4B ). Taken together, these data suggest that Set1 is required in postmitotic MB neurons for normal memory.
We also assessed sleep, activity, and baseline courtship behavior in MB-specific Set1 knockdown flies. There was a small but significant (p= 0.03) reduction in sleep for Set1 RNAi1 , however, this reduction was not observed for Set1 RNAi2 (Supplementary Figure 4A imaged whole mount adult brains using confocal microscopy. We did not observe gross morphological defects in larval, pupal, or adult stages in Set1 knockdown flies (Supplementary Figure   4D ). Thus, Set1 appears to be dispensable in postmitotic neurons for normal MB morphogenesis.
Taken together with our behavior data, this suggests that Set1 is required for normal functioning of adult MB neurons during the formation of short-and long-term courtship memory.
The presented clinical and molecular data strongly support the conclusion that heterozygous LoF of human SETD1A causes a novel defined neurodevelopmental disorder. The core features associated with SETD1A haploinsufficiency include global developmental delay or intellectual disability, subtle facial dysmorphisms, and psychiatric problems. In previous studies heterozygous LoF variants in SETD1A were observed in individuals with schizophrenia 14 . Therefore, in this study we investigated the behavioral and psychiatric abnormalities that have been observed in our predominantly pediatric cohort. No patient had a previous diagnosis of schizophrenia, but this is expected because of the relatively young age of the patients. With the exception of the very rare childhood onset forms 47 , schizophrenia rarely manifests so early 48 . Interestingly, an adolescent and a young adult from our cohort (age 16 years and 23 years) showed signs that could be linked with schizophrenia or bipolar disorder, though they had no formal psychiatric diagnosis. Combined with the previous reports, this indicates that the germline SETD1A variants might predispose individuals to severe neuropsychiatric pathologies later in life. Therefore, this patient group should be carefully monitored for suggestive psychiatric symptoms.
It is notable that in humans all six H3K4 methyltransferases, SETD1A/B, KMT2A/B, and KMT2C/D have been implicated in neurodevelopmental disorders, though it is also clear that all KMT2 members have distinct molecular functions 30, 41, [49] [50] [51] . However, there is an interesting overlap between these chromatin modulating genes and their respective phenotypes (with developmental delay/intellectual disability at its core), based on their shared underlying disease mechanisms 52 .
In the 15 patients investigated in this study we identified 14 different SETD1A variants. The majority of these were LoF nonsense or frameshift variants, however a twice recurrent splice-site variant and a missense variant were also identified in the catalytic SET domain, which is required for methyltransferase activity. Previously, SETD1A-mediated H3K4 methylation was shown to be important for protecting against DNA damage at stalled replication forks 12 . We used this cellular phenotype as an output to test the functional effects of clinical SETD1A variants in three patient derived lymphoblastoid cell lines. Interestingly, cell lines with heterozygous LoF alleles (p.Gly535Alafs*12, and c.4582-2_4582delAG) showed high levels of nascent DNA degradation, at a level of severity that was similar to that observed in cell lines treated with SETD1A siRNA 12 .
Moreover, cells with the heterozygous missense variant (p.Tyr1499Asp) also showed a similar phenotype, thus supporting the pathogenicity of this variant. Taken together, these results suggest that SETD1A-associated clinical features likely result from partial loss of SETD1A methyltransferase activity.
In two unrelated patients we found the same de novo two base pair (bp) deletion (c.4582-2_4582- Figure 5 ). This suggests a significant role for this particular exon for the functionality of the N-SET domain, which is partly encoded by this exon.
Our RNA analyses surprisingly revealed that exon skipping did not occur, but the two bp deletion rather resulted in retention of intron 15. In the three other studies describing patients with variants in SETD1A, the exact same variant was found in 7 out of the total of 16 patients 14, 23, 24 . In addition, this variant occurs at a frequency of 8.071e -6 in the GnomAD database 40 . Authors from the previous studies did not speculate on this seemingly unlikely recurrence of the two bp deletion 14, 24 . However, it raises questions on the frequency and etiology of occurrence of this variant. It is possible that the mutation rate in this genomic area, or specifically around these two base pairs, is relatively high. This may be due to the surrounding sequence homology or the epigenetic state of the DNA, which in turn may allow this deletion to occur. Another explanation might be ascertainment bias, due to the possibility that the specific variant gives rise to ID and/or schizophrenia. Whereas other SETD1A variants might also be causative for other phenotypes and therefore would not be included in studies focusing on ID or schizophrenia. In addition, it might be that the c.4582-2_4582-1delAG variant is a so-called selfish mutation. Certain DNA variants that can occur over time in spermatogonial cells can lead to clonal expansion (such as the c.755C>G variant in the FGFR2 gene in the case of Apert syndrome). This can culminate to the passing of that certain variant from father to child, leading to a higher prevalence in the patient group. Typically these variants are 'Gain of Function' (GoF) 55, 56 , so in our cohort this does not seem to be the case.
The Drosophila SETD1A Orthologue is required in Postmitotic Neurons for Memory SETD1A and other COMPASS components have been implicated in the regulation of cell proliferation and differentiation. However, there is little evidence that these processes solely underlie the clinical features that we describe in SETD1A haploinsufficiency, as the cognitive deficits and facial dysmorphisms reported in our cohort are mild. Defects in proliferation or differentiation of neural progenitors might have more dramatic effects on structure and function of the brain, and cause profound cognitive deficits or lethality. In line with this, we identified a role for Drosophila Set1 in the regulation of memory formation in postmitotic neurons. This finding is consistent with studies demonstrating a role for different histone modifications in the epigenetic regulation of memory 57 .
For example, H3K4me3 is dynamically regulated in the rodent hippocampus during memory formation and Setd1a +/mice exhibit deficits in working memory 58, 59 . Several other H3K4 methyltransferases have also been implicated in memory, including mouse Kmt2a/Kmt2b and The specific role of H3K4 methylation in memory formation is not known. Set1-related proteins might regulate several different aspects of postmitotic neuronal biology that could all impact memory. For example, we have shown here that patient cell lines have elevated damage in nascent DNA. Terminally differentiated neurons are often exposed to DNA damage and an efficient response is critical, as these neurons are not easily replaced. Additionally, the Set1 protein family might be important for maintaining the cell-type specific gene expression programs that would be critical for normal functioning of memory neurons. Set1-related proteins might also be required for induction of learning-induced gene expression changes that are known to be required for long-term memory formation.
Despite the lack of a precise pathogenetic mechanism, the finding that Set1 regulates memory post (B) Patient-derived LCL were exposed to 2 mM HU for 24 h, WCE prepared and analysed by immunoblotting using the denoted antibodies. Data is representative of n=2 independent experiments. * Denotes a non-specific band.
(C) Patient-derived LCL were sequentially pulsed for 20 min each with CldU and IdU, and exposed to 4 mM HU for 5 h (as in the schema). DNA was visualised with antibodies to CldU and IdU, and tract length was calculated. Plots denote the average ratios of IdU:CldU label length from three independent experiments. Red lines indicate mean ratios. Data represents pooled values from n=3 experiments, representative images are shown. As information on the different features was not always applicable or known for each patient, the denominator in the 'Frequency' column is different for different clinical characteristics.