# Dissecting the 22q13 region to explore the genetic and phenotypic diversity of patients with Phelan-McDermid syndrome

Aline Vitrac, Claire Leblond, Thomas Rolland, Freddy Cliquet, Mathieu Alexandre, Anna Maruani, Richard Delorme, Michael Schön, Andreas Grabrucker, Conny van Ravenswaaij-Arts, et al.

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## To cite this version:

Aline Vitrac, Claire Leblond, Thomas Rolland, Freddy Cliquet, Mathieu Alexandre, et al. Dissecting the 22q13 region to explore the genetic and phenotypic diversity of patients with Phelan-McDermid syndrome. European Journal of Medical Genetics, 2023, 66 (5), pp.104732. 10.1016/j.ejmg.2023.104732. pasteur-04119437

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# Dissecting the 22q13 region to explore the genetic and phenotypic diversity of patients with Phelan-McDermid syndrome

Aline Vitrac<sup>a, **</sup>, Claire S. Leblond<sup>a</sup>, Thomas Rolland<sup>a</sup>, Freddy Cliquet<sup>a</sup>, Alexandre Mathieu<sup>a</sup>,
Anna Maruani<sup>b</sup>, Richard Delorme<sup>b</sup>, Michael Schön<sup>c</sup>, Andreas M. Grabrucker<sup>d, e, f</sup>,
Conny van Ravenswaaij-Arts<sup>g</sup>, Katy Phelan<sup>h</sup>, Anne-Claude Tabet<sup>i</sup>, Thomas Bourgeron<sup>a, *</sup>

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

Handling Editor: A. Verloes

**Keywords:**

Phelan-McDermid syndrome
Neurodevelopmental disorders
Autism
Epilepsy
Brain development
SHANK3

## ABSTRACT

SHANK3-related Phelan-McDermid syndrome (PMS) is caused by a loss of the distal part of chromosome 22, including SHANK3, or by a pathological SHANK3 variant. There is an important genetic and phenotypic diversity among patients who can present with developmental delay, language impairments, autism, epilepsy, and other symptoms. SHANK3, encoding a synaptic scaffolding protein, is deleted in the majority of patients with PMS and is considered a major gene involved in the neurological impairments of the patients. However, differences in deletion size can influence clinical features, and in some rare cases, deletions at the 22q13 locus in individuals with SHANK3-unrelated PMS do not encompass SHANK3. These individuals with SHANK3-unrelated PMS still display a PMS-like phenotype. This suggests the participation of other 22q13 genes in the pathogenesis of PMS. Here, we review the biological function and potential implication in PMS symptoms of 110 genes located in the 22q13 region, focusing on 35 genes with evidence for association with neurodevelopmental disorders, including 13 genes for epilepsy and 11 genes for microcephaly and/or macrocephaly. Our review is restricted to the 22q13 region, but future large-scale studies using whole genome sequencing and deep-phenotyping are warranted to develop predictive models of clinical trajectories and to target specific medical and educational care for each individual with PMS.

## 1. Introduction

Phelan-McDermid syndrome (PMS) is a severe neurodevelopmental disorder (NDD) defined by chromosomal rearrangements impacting the 22q13 region of the long arm of chromosome 22, resulting in a loss of genetic material. Various mechanisms have been reported, including simple small to large deletions, unbalanced translocations, complex genomic rearrangement of the distal part of the long arm of chromosome 22, and the formation of a ring chromosome (Koza et al., this issue). These events arise mainly *de novo*, but germinal mosaicism, balanced

chromosomal rearrangements, or rare cases of inherited deletions from the parents are also observed (Bonaglia et al., 2011, 2020; Denayer et al., 2012; Tabet et al., 2017). In individuals with PMS, the deletion size ranges from less than 100 kb to over 9 Mb. For terminal deletions, the most proximal breakpoint in the long arm of chromosome 22 has been mapped in the 22q13.2 cytogenetic region (Sarasua et al., 2014a).

For most individuals with PMS, deletions include the terminal 22q13 region and cover the entire SHANK3 gene. Interstitial deletions overlapping exons of SHANK3 and deleterious point mutations in the coding sequence of SHANK3 are also observed (Durand et al., 2007; Tabet et al.,

<footnote><sup>a</sup> Génétique Humaine et Fonctions Cognitives, Institut Pasteur, UMR3571 CNRS, Université de Paris Cité, IUF, 75015, Paris, France</footnote>

<footnote><sup>b</sup> Department of Child and Adolescent Psychiatry, Hôpital Robert Debré, APHP, Paris, France</footnote>

<footnote><sup>c</sup> Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany</footnote>

<footnote><sup>d</sup> Bernal Institute, University of Limerick, Limerick, Ireland</footnote>

<footnote><sup>e</sup> Dept. of Biological Sciences, University of Limerick, Limerick, Ireland</footnote>

<footnote><sup>f</sup> Health Research Institute HRI, University of Limerick, Limerick, Ireland</footnote>

<footnote><sup>g</sup> University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands</footnote>

<footnote><sup>h</sup> Genetics Laboratory, Florida Cancer Specialists & Research Institute, Fort Myers, FL, 33916, USA</footnote>

<footnote><sup>i</sup> Department of Genetics, Hôpital Robert Debré, APHP, Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: vitrac.aline@gmail.com (A. Vitrac), thomas.bourgeron@pasteur.fr (T. Bourgeron).</footnote>
2017; De Rubeis et al., 2018). These observations suggest that *SHANK3* is the main contributor to the PMS phenotype. However, in some patients with clinical features similar to PMS, interstitial deletions do not affect the protein-coding regions of *SHANK3* (Disciglio et al., 2014; Simenson et al., 2014; Palumbo et al., 2018; Li et al., 2020; Wilson et al., 2008). A new classification system was recently proposed to differentiate patients with *SHANK3*-related PMS and *SHANK3*-unrelated PMS (Phelan et al., 2022).

The core phenotypes of PMS are absent or delayed speech, intellectual disability (ID), neonatal hypotonia, global developmental delay, minor morphological features such as large and fleshy hands, and multiple comorbidities with variable expressivity (Phelan and McDermid 2011; Schön et al., this issue). Autism spectrum disorder (ASD) is a prevalent phenotype of PMS, as observed in up to 70–80% of the patients (Soorya et al., 2013; Xu et al., 2020, Schön et al., this issue, van Balkom et al., this issue). Other impairments include epilepsy, sleep disturbance, gastrointestinal abnormalities, lymphedema, renal malformations, and feeding problems (Phelan and McDermid, 2011; Schön et al., this issue). In addition, sensory functioning is often impacted in individuals with PMS, which leads to visual and hearing impairments, reduced pain perception, heat regulation disorder and proprioceptive and vestibular problems (Walinga et al., this issue). Patients with PMS may also exhibit abnormal head growth with microcephaly and macrocephaly in 16% and 17% of cases, respectively (Schön et al., this issue).

In the last decade, a number of studies aiming to understand better the clinical heterogeneity among individuals with PMS were performed in different countries (Soorya et al., 2013; Sarasua et al., 2014b; Tabet et al., 2017; Samogy-Costa et al., 2019; Xu et al., 2020; Nevado et al., 2022). Such investigations found correlations between the size of the deletion and the presence or severity of the PMS symptoms. This has led to the discovery of specific genomic regions that could be associated with an increased risk of absence of speech, gastroesophageal reflux, ophthalmic features, macrocephaly, or epilepsy, among others (Table 1; Sarasua et al., 2014a; Tabet et al., 2017; Jain et al., 2022). In this article, we review the function of 35 genes throughout the 22q13 region deleted in individuals with PMS to provide a state-of-the-art list of the genetic factors at 22q13 that could influence the clinical trajectories of patients with PMS.

## 2. Definition of the 22q13 region, PMS deletions and gene lists

The region of interest was established, using the genomic coordinates of published deletions carried by individuals with PMS (Sarasua et al., 2014a), between the most proximal breakpoint reported so far (hg19; chr22:41,770,054) and the distal part of the chromosome 22 (hg19; chr22:51,304,566). The region contains four cytogenetic subregions of the 22q13 region (22q13.2, 22q13.31, 22q13.32, and 22q13.33) and 110 protein-coding genes, as shown in Fig. 1A. In addition, there are 175 genes encoding non-protein coding RNA within this region.

In order to have a comprehensive view of the deletion size diversity of the 22q13 region in PMS, we integrated the genetic data of 57

individuals with PMS from our French study with the data of 325 patients registered in the PMSF DataHub which has been initiated thanks to the PMS Foundation (https://pmsf.org/). Genetic data were downloaded from the PMSF registry in August 2022 and phenotypic data corresponding to patients included in this study are also available upon request to the PMSF. Interstitial deletions reported in 19 individuals with PMS from literature and two individuals with PMS from our French study are presented separately (Supplemental Fig. 1). As illustrated in Fig. 1B, the breakpoint positions of deletions are dispersed uniformly along the distal part of chromosome 22, resulting in a continuous range of deletion sizes. However, we identified three major groups of individuals with PMS carrying a 22q13 deletion, with distinct numbers of deleted genes: one group of individuals with less than four deleted genes representing 24% of the patients, one group with 35–37 deleted genes representing 24% of the patients and one group with more than 45 deleted genes representing more than 30% of the individuals with PMS (Fig. 1C). A genomic region (chr22:44,850,001-45,630,709 in hg19) appears frequently deleted in individuals with PMS carrying interstitial deletions and contains seven genes (Supplemental Fig. 1).

In the following sections, we describe specific genes located within the 22q13 region that could contribute to the neurological symptoms of individuals with PMS. For this purpose, we used GeneTrek (https://gnetrek.pasteur.fr/, Leblond et al., 2021), which gathers different databases related to NDD. Genes are listed in three non-overlapping categories depending on their association level with NDD. The high confidence NDD (HC-NDD) genes correspond to 1,724 genes, which are considered as robustly associated with NDD by at least one of five major sources: SPARK gene list (https://sparkforautism.org/), SFARI (https://www.sfari.org/), SysNDD (https://sysndd.dbmr.unibe.ch/), DDG2P (https://www.deciphergenomics.org/ddd/ddgenes) and DBD (https://dbd.geisingeradmi.org/). The second category includes 5,727 candidate NDD genes, which contain genes with a lower level of confidence for their involvement in NDD (2,143), genes associated with epilepsy, microcephaly or macrocephaly not included in the HC-NDD list, and genes specifically expressed in the foetal and/or adult human brain and/or intolerant to loss of function (LoF) genetic variants. Finally, 12, 381 genes are neither HC-NDD nor candidate NDD genes. More details concerning the inclusion criteria for the HC-NDD and the candidate NDD gene lists are provided in Supplemental Figs. 2 and 3. Other information is present on GeneTrek, including the association with epilepsy and micro/macrocephaly. Intolerance to LoF mutations is based on pLI (probability of being LoF intolerant) and LOEUF (LoF observed/expected upper bound fraction). Both metrics were extracted from gnomAD. The LOEUF is a recent metric that considers the length of the gene and the number of samples. It allows estimating the tolerance to LoF mutations with more continuous values than the pLI. A gene with a LOEUF<0.35 is considered intolerant to LoF (Karczewski et al., 2020). For brain expression analyses, we used data from BrainSpan and Gtex (https://www.brainspan.org, https://www.gtxportal.org/home/).

<table><caption>**Table 1**
Frequency of PMS clinical features and chromosome 22q13 associated genomic positions.</caption><thead><tr><th>Clinical features</th><th>Frequency</th><th>Genomic region associated (hg 19)</th><th>Relative risk (prevalence in carriers of deletions in the associated region)</th><th>Reference</th></tr></thead><tbody><tr><td>Neonatal hypotonia</td><td>80% (n = 60)</td><td>[45.5 Mb–50.6 Mb]</td><td>increased risk</td><td>Sarasua et al. (2014a)</td></tr><tr><td rowspan="2">Absence of speech</td><td>49% (n = 73)</td><td>[42.6 Mb–46.3 Mb]</td><td>increased risk (80%)</td><td>Tabet et al. (2017)</td></tr><tr><td>69% (n = 35)</td><td>[43.6 Mb–47.1 Mb]</td><td>increased risk</td><td>Sarasua et al. (2014a)</td></tr><tr><td>Seizures</td><td>48% (n = 46)</td><td>[45.6 Mb–48.5 Mb]</td><td>increased risk</td><td>Jain et al. (2022)</td></tr><tr><td>Macrocephaly</td><td>23% (n = 47)</td><td>[46.1 Mb–48.2 Mb]</td><td>increased risk</td><td>Sarasua et al. (2014a)</td></tr><tr><td>Ophthalmic feature</td><td>30% (n = 72)</td><td>[42.25 Mb–44.6 Mb]</td><td>increased risk (70%)</td><td>Tabet et al. (2017)</td></tr><tr><td>Gastrointestinal reflux</td><td>19% (n = 72)</td><td>[48.9 Mb–49.9 Mb]</td><td>increased risk (70%)</td><td>Tabet et al. (2017)</td></tr></tbody></table>
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Ophtalmic features (Tabet et al 2017)

Macrocephaly (Sarasua et al 2014b)

Gastroesophageal reflux (Tabet et al 2017)

Seizures (Jain et al 2022)

Absence of speech (Sarasua et al 2014b)

Absence of speech (Tabet et al 2017)

Hypotonia (Sarasua et al 2014b)

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

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(caption on next page)
**Fig. 1.** **HC-NDD and candidate NDD genes encompassing the regions deleted in patients with PMS.** A. Representation of the 22q13 region with all deleted genes displayed in black (n = 110). HC-NDD are genes listed in SFARI I, SPARK, primary SysNDD, DDG2P Brain, or Cognition with a confirmed status and the Tier1 or autosomal recessive inheritance list on Developmental Brain Disorder (DBD). Candidate NDD genes are (i) genes with a lower level of confidence for their involvement in NDD, (ii) genes associated with epilepsy, microcephaly, or macrocephaly, not in the HC-NDD list, and genes specifically expressed in the foetal and/or adult human brain and/or intolerant to LoF genetic variants. HC-NDD genes with a dominant or recessive inheritance are displayed respectively in red (n = 4) and pink (n = 8). Candidate NDD genes are represented in blue (n = 23). Regions associated with ophthalmic features, macrocephaly, seizures, absence of speech, gastroesophageal reflux and hypotonia are also illustrated. B. Terminal deletions of patients included in this study are shown by horizontal lines. For patients of the PMSF DataHub (n = 325), genomic coordinates were determined through array technologies and were given either in hg17, hg18, hg19, or hg38. Minimum genomic coordinates were all converted in hg19 using the liftOver package of R (version 1.20.0). Only patients with array data and both start and end genomic coordinates available are shown. We excluded duplicated individuals from France and individuals with multiple events in the region. For patients of our cohort (n = 57), genomic coordinates were validated by CRAM visualisation obtained after whole genome sequencing. A scale representing the number of individuals deleted for the region is shown at the top of the figure. C. Distribution of individuals with a 22q13 deletion in function to the number of deleted genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

**3. The 22q13 region and the HC-NDD and candidate NDD genes**

Among 110 protein-coding genes in the 22q13 region, 12 (10.9%) are HC-NDD genes, including four with a dominant inheritance: ACO2, SCO2, TCF20, and SHANK3. A total of 21 candidate NDD genes could also participate in the PMS phenotype and account for 19% of the

protein-coding genes encompassing the 22q13 region. RNU12 and LINC00634, two genes encoding non-protein-coding RNA, also belong to this category. More than half of individuals with PMS carry deletions encompassing two HC-NDD genes with dominant inheritance (SHANK3 and SCO2) and five HC-NDD genes with recessive inheritance (ARSA, CHKB, TUBGCP6, MLC1, and ALG12; Fig. 1C).

<figure><img src="image_8.png" /></figure>

**Fig. 2.** **Characterisation of HC-NDD and candidate NDD genes in the 22q13 region.** Each HC-NDD dominant (red) or recessive (pink) and candidate NDD (blue) genes are represented on the circle heatmap. Genes for epilepsy (dark green), microcephaly (yellow), macrocephaly (orange), or both micro/macrocephaly (brown) are taken from HPO (https://hpo.jax.org/app/). Information on tolerance to LoF mutations is displayed in grey in the LOEUF section, with light grey, and dark grey respectively, representing genes tolerant (LOEUF$\ge$0.35) or intolerant (LOEUF<0.35) to LoF mutations. The last section shows 1) a score indicating which genes are more expressed in the pre-birth period (cyan) or the post-birth period (green), 2) a score measuring the brain specificity (purple), and 3) the genes listed in SynGO (light brown). Scores to evaluate brain specificity and temporal expression were calculated using Tau, a metric widely used to evaluate tissue specificity of a gene (Kryuchkova-Mostacci and Robinson-Rechavi, 2016). The plot was constructed with the R package (version 0.4.15) circlize (Gu et al., 2014). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
In total, 15 genes of the 35 HC-NDD or candidate NDD genes located in the 22q13 region are predicted to be intolerant to LoF mutations (LOEUF <0.35; Fig. 2). Six HC-NDD or candidate NDD genes (MLC1, PNPLA5, SULT4A1, MPPED1, LINC00634, and SHISA8) appear to be specifically expressed in the brain (Fig. 2). Several genes are also more expressed during the pre-birth period (e.g., PLXNB2, BRD1, FBLN1, MPPED1), while others are more expressed during the post-birth period (e.g., SHANK3, MLC1, SULT4A1; Fig. 2).

Below is a description of the four HC-NDD genes with a dominant inheritance at 22q13.

**SHANK3** is deleted or carries point mutations in all individuals with PMS presented here. In the PMS registry, **SHANK3** point mutations represent 12% of individuals with available genetic data. **SHANK3** has been identified as a major gene associated with autism in up to 2% of autistic individuals with ID (Durand et al., 2007; Leblond et al., 2014). **SHANK3** encodes a scaffolding protein that makes the bridge between the actin cytoskeleton and the proteins at the membrane at glutamatergic postsynapses (Boeckers et al., 2002). Many **Shank3** mouse models deleting all or specific isoforms have been generated to study the difference in the cellular phenotype, brain circuit, and neuronal activity compared to wild-type mice. **Shank3** deficient mice are characterised by motor dysfunction, reduced cognitive functions, impaired synaptic transmission and plasticity, and abnormal dendritic spine morphology and density (Delling and Boeckers, 2021). Mutations in **SHANK3** were also observed in other psychiatric disorders, such as bipolar disorders and schizophrenia (Gauthier et al., 2010; de Sena Cortabitarte et al., 2017).

**SCO2** is deleted in 74% of individuals with PMS and codes for a protein located at the inner membrane of the mitochondria, where it plays a significant role in energy production. **SCO2** encodes a nuclear subunit of the cytochrome c oxidase (COX), also known as the complex IV of the mitochondrial chain that is involved in aerobic ATP production. Autosomal recessive mutations in proteins forming this complex represent about 15% of cases of Leigh syndrome, a severe neurological disorder characterised by a loss of psychomotor skills (Lake et al., 2016; Bakare et al., 2021). Other studies highlighted an association of **SCO2** dominant mutations with severe forms of myopia in which the dioptic power is greater than six (Tran-Viet et al., 2013; Wakazono et al., 2016). Mutations leading to this ophthalmic dysfunction are either LoF or missense variant (Tran-Viet et al., 2013; Wakazono et al., 2016). Homozygous and compound heterozygous mutations in **SCO2** have also been reported in individuals with fatal infantile cardioencephalomyopathy (Tay et al., 2004).

**TCF20** is deleted in 5% of individuals with PMS and codes for a nuclear protein, acting as a transcriptional coactivator. It regulates the expression of other transcription factors and MMP3, an enzyme implicated in extracellular matrix protein degradation. **TCF20** is associated with a developmental delay with variable intellectual impairment and autism (Lévy et al., 2022). Three de novo **TCF20** mutations were identified in autistic individuals, and an inversion impacting **TCF20** was reported in two brothers with autism and mild ID (Babbs et al., 2014). In line with these results, mice carrying a deletion of *Tcf20* show alteration of the cortical neurogenesis during embryogenesis in addition to impairments in social communication and interaction co-occurring with excessive repetitive behaviour (Feng et al., 2020).

The mitochondrial aconitase enzyme encoded by **ACO2** is deleted in very few patients who carry very large 22q13 deletions (>9 Mb). It catalyses the second reaction of the Krebs cycle consisting of the isomerisation of citrate to isocitrate. Homozygous or compound heterozygous variants in **ACO2** lead to infantile cerebellar retinal degeneration, a rare recessive neurodegenerative disorder (ICRD; Spiegel et al., 2012; Sharkia et al., 2019). Patients with ICRD present development delay, ophthalmological defects, atrophy of the cerebellum, epilepsy, and microcephaly (Spiegel et al., 2012; Sharkia et al., 2019). The severity of ophthalmological impairments correlates with a deficit in aconitase activity (Metodiev et al., 2014; Sharkia et al., 2019). A large proportion

of patients are also nonverbal and nonambulatory. When isolated, optic neuropathies can be due either to dominant or recessive **ACO2** mutations (Guehlouz et al., 2021). Other disorders are related to mutations in **ACO2**, such as hereditary spastic paraplegia and optic atrophy 9 (Marelli et al., 2018; Tozawa et al., 2021). In the case of optic atrophy, there is both dominant and recessive inheritance (Charif et al., 2021).

Besides the four dominant HC-NDD genes presented above, 8 HC-NDD recessive genes and 23 candidate NDD genes might also be involved in various disorders affecting the brain or other organs and could contribute to many clinical features of individuals with PMS, as shown in Table 2. Since epilepsy, microcephaly, and macrocephaly are neurological-related symptoms reported in PMS, we used the human phenotype ontology (HPO) terms to select genes located in the 22q13 region associated with these phenotypes. More details about their biological function and involvement in epilepsy, microcephaly, and macrocephaly are presented below.

## 4. The 22q13 region and epilepsy

Epilepsy is a common comorbidity in individuals with PMS, with a frequency that fluctuates between 17% and 70% and a lifelong increase (Holder et Quach, 2016, De Coo et al., this issue; Schön et al., this issue). The type of epilepsy in PMS patients varies between febrile, focal, atonic, tonic-clonic, tonic, and myoclonic seizures (Jain et al., 2022). The seizure frequency and trigger are also variable among patients (Jain et al., 2022). Why some individuals with PMS develop epilepsy and others do not remains unclear. Contrary to other comorbidities, such as the absence of speech, a number of studies failed to find a strong correlation between epilepsy prevalence and deletion sizes (Reierson et al., 2017; Sarasua et al., 2014b; Tabet et al., 2017). However, one study found an increasing proportion of patients with PMS and epilepsy carrying large deletions >4 Mb (Jain et al., 2022). Interestingly, the parental origin of the affected chromosome has been suggested to play a role in seizure development, with a higher risk for deletion occurring on the maternal chromosome (Sarasua et al., 2014b). Better knowledge of the genetic factors involved in epilepsy is important for treating the seizures occurring in individuals with PMS (Musto et al., 2020).

Based on HPO terms, we established a list of 1,736 candidate genes for epilepsy. In total, 13 of them (SHANK3, ARSA, CHKB, SCO2, TUBGCP6, MLC1, ALG12, ATXN10, FBLN1, CYB5R3, TCF20, NAGA and ACO2) map to the 22q13 region (Fig. 2).

Manifestations of at least one epileptic event during a lifetime have been reported in two patients with a frameshift mutation affecting **SHANK3** (Holder and Quach, 2016). Both patients were 14 years old and were affected by atypical absence epilepsy. The first seizures of patient 1 appeared at 7 years of age and occurred multiple times per week with a duration of 3–5 min. Onset of epilepsy was at 14 years of age for patient 2, and seizures spanned 3 min. In another study, among 15 autistic individuals carrying a genetic variant in **SHANK3**, 6 presented with epilepsy (Leblond et al., 2014). Similarly, 29% of a cohort with 17 carriers of a LoF mutation in **SHANK3** also developed epilepsy (De Rubeis et al., 2018). Despite the large number of mutant mice for **Shank3**, epilepsy has been barely observed in this model (Peça et al., 2011). In contrast, mice overexpressing **Shank3** display spontaneous seizures, as shown in two different studies (Han et al., 2013; Jin et al., 2018).

**ARSA** (deleted in 77% of PMS individuals) encodes an enzyme expressed in lysosomes implicated in the degradation of sulfatide, a lipid located in the nervous system's myelin. Low activity of ARSA leads to metachromatic leukodystrophy (MLD), a rare neurodegenerative disorder characterised by progressive motor and cognitive impairments. This disorder is mainly caused by autosomal recessive mutations in ARSA. Depending on the level of ARSA activity, the onset of MLD varies between adult (15–20% of cases), juvenile (20–30% of cases), and late infantile (50–60% of cases), which is the most common and severe form (Shaimardanova et al., 2020). Epilepsy has been reported in two
<caption>**Table 2**</caption>

**Genes in the 22q13 regions and their associated diseases.** Genes and associated diseases were extracted from the OMIM morbid map. OMIM ID is given for each phenotype. HC-NDD genes with a dominant or recessive inheritance are displayed respectively in red and pink. Candidate NDD genes and other genes are displayed respectively in blue and black.

<table><thead><tr><th>Gene</th><th>morbid MAP, OMIM id</th></tr></thead><tbody><tr><td colspan="2">From 40 Mb to 42 Mb</td></tr><tr><td rowspan="2">AC02</td><td>Infantile cerebellar-retinal degeneration, 614559</td></tr><tr><td>Orofacial cleft 10, 613705</td></tr><tr><td colspan="2">From 42 Mb to 44 Mb</td></tr><tr><td>MEI1</td><td>Hydatidiform mole, recurrent, 3, 618431</td></tr><tr><td>CCDC134</td><td>Osteogenesis imperfecta, type XXII, 619795</td></tr><tr><td>TNFRSF13C</td><td>Immunodeficiency, common variable, 4, 613494</td></tr><tr><td rowspan="3">NAGA</td><td>Kanzaki disease, 609242</td></tr><tr><td>Schindler disease, type I and III, 609241</td></tr><tr><td>Schindler disease, type III, 609241</td></tr><tr><td>NDUFA6</td><td>Mitochondrial complex I deficiency, nuclear type 33, 618253</td></tr><tr><td rowspan="2">CYP2D6</td><td>Codeine sensitivity, 608902</td></tr><tr><td>Debrisoquine sensitivity, 608902</td></tr><tr><td>TCF20</td><td>Developmental delay with variable intellectual impairment and behavioural abnormalities, 618430</td></tr><tr><td>RRP7A</td><td>Microcephaly 28, primary, autosomal recessive, 619453</td></tr><tr><td rowspan="2">CYB5R3</td><td>Methemoglobinemia, type I, 250800</td></tr><tr><td>Methemoglobinemia, type II, 250800</td></tr><tr><td rowspan="3">A4GALT</td><td>NOR polyagglutination syndrome, 111400</td></tr><tr><td>Blood group, P1Pk system, P(2) phenotype, 111400</td></tr><tr><td>Blood group, P1Pk system, p phenotype, 111400</td></tr><tr><td colspan="2">From 44 Mb to 46 Mb</td></tr><tr><td>FBLN1</td><td>Synpolydactyly, 3/3'4, associated with metacarpal and metatarsal synostoses, 608180</td></tr><tr><td colspan="2">From 46 Mb to 48 Mb</td></tr><tr><td>ATXN10</td><td>Spinocerebellar ataxia 10, 603516</td></tr><tr><td>PPARA</td><td>Hyperapobetalipoproteinemia, susceptibility to</td></tr><tr><td rowspan="2">TRMU</td><td>Liver failure, transient infantile, 613070</td></tr><tr><td>Deafness, mitochondrial, modifier of, 580000</td></tr><tr><td>CELSR1</td><td>Lymphatic malformation 9, 619319</td></tr><tr><td colspan="2">From 48 Mb to 50 Mb</td></tr><tr><td colspan="2">From 50 Mb to 52 Mb</td></tr><tr><td>ALG12</td><td>Congenital disorder of glycosylation, type Ig, 607143</td></tr><tr><td>MLC1</td><td>Megalencephalic leukoencephalopathy with subcortical cysts, 604004</td></tr><tr><td>MOV10L1</td><td>Spermatogenic failure 73, 619878</td></tr><tr><td>TUBGCP6</td><td>Microcephaly and chorioretinopathy, autosomal recessive, 1, 251270</td></tr><tr><td>SBF1</td><td>Charcot-Marie-Tooth disease, type 4B3, 615284</td></tr><tr><td rowspan="2">SCO2</td><td>Mitochondrial complex IV deficiency, nuclear type 2, 604377</td></tr><tr><td>Myopia 6, 608908</td></tr><tr><td>TYMP</td><td>Mitochondrial DNA depletion syndrome 1 (MNGIE type), 603041</td></tr><tr><td>CHKB</td><td>Muscular dystrophy, congenital, megaconial type, 602541</td></tr><tr><td>ARSA</td><td>Metachromatic leukodystrophy, 250100</td></tr><tr><td rowspan="2">SHANK3</td><td>Phelan-McDermid syndrome, 606232</td></tr><tr><td>Schizophrenia 15, 613950</td></tr><tr><td>ACR</td><td>Male infertility due to acrosin deficiency</td></tr></tbody></table>
independent cases of patients with juvenile MLD and recessive variants in *ARSA* (Kang et al., 2010; Barboura et al., 2022).

**CHKB** (deleted in 75% of PMS individuals) is involved in the metabolism of phospholipids and is the causative gene of congenital muscular dystrophy. Clinical features of 15 patients carrying *CHKB* mutations between 38 days of age and 17 years of age were analysed. Among them, three individuals carrying recessive variants in *CHKB* were subject to a single epilepsy history (Haliloglu et al., 2015).

Epilepsy has also been observed in three children with hypertrophic cardiomyopathy and COX deficiency (Jaksch et al., 2000). They belong to two unrelated families where pathogenic mutations in *SCO2* segregate. The three patients carried compound heterozygous genetic variants and shared one predicted deleterious missense variant E140K (Jaksch et al., 2000).

Epilepsy is a frequent comorbid phenotype of megalencephalic leuкоencephalopathy with subcortical cysts (MLC), which is observed in 75% of cases with **MLC1** homozygous recessive mutations. This gene (deleted in 68% of PMS individuals) codes for a protein specifically expressed in astrocytes (Leegwater et al., 2001; Yalçinkaya et al., 2003). Epilepsy severity varies, ranging from mild to severe cases with status epilepticus. In general, antiepileptic treatments efficiently control mild epilepsy in MLC patients (van der Knaap et al., 2012). In addition, *Mlc1* deficient mice exhibit signs of higher epilepsy susceptibility, including hindlimb clasping, abnormal electrical activity, and lower seizure threshold. Induced epilepsy is also more severe in these mice than in wild-type littermates (Dubey et al., 2018).

**ALG12** (deleted in 65% of PMS individuals) is one of the causative genes of a multi-system disorder called congenital disorder of glycosylation (CGC) type Ig. The encoded protein is a reticulum enzyme with transferase activity essential for protein glycosylation. Other types of CGC exist and imply different genes with gene-specific phenotypes (Haeuptle and Hennet, 2009). Concerning ALG12-CGC, the disorder is inherited with a recessive model, and some patients affected by this disorder also display epilepsy (Tahata et al., 2019).

Generalised motor epilepsy is recurrently observed in spinocerebellar ataxia type 10, whose origin is due to the expansion of a 5 nucleotides pattern (ATCCT) located in intron 9 of *ATXN10* (deleted in 30% of PMS individuals; Rasmussen et al., 2001). There is an increased risk of developing epilepsy when ACTTC repeats are interrupted by a heptanucleotide pattern (ATTTTCT or ATATTCT) or when a first-degree relative also has epilepsy (McFarland et al., 2014). However, in the case of PMS, the 22q13 deletion leads to a loss of one copy of *ATXN10*, and there is no report of epilepsy in patients with *ATXN10* haploinsufficiency.

**CYB5R3** (deleted in 8% of PMS individuals) codes for two isoenzymes. Isoform 1 is a soluble protein expressed specifically in erythrocytes, whereas isoform 2 is expressed in all tissues and binds to the outer mitochondrial membrane, the endoplasmic reticulum, and the plasma membrane. Mutations in *CYB5R3* cause a rare disease called recessive hereditary methemoglobinemia (RHM) which exists in two forms: RHM type I and RHM type II. RHM type II is the most severe form, with a deficiency in the membrane-anchored isoform, which is essential for various metabolic transformations. RHM type II results in diverse neurological impairments, including epilepsy in 16% of the patients, the majority being pharmaco-resistant (Nicita et al., 2022).

The presence of epilepsy in carriers of variants in *TCF20* has been evaluated in four studies (Lelieveld et al., 2016; Schäfgen et al., 2016; Torti et al., 2019; Vetrini et al., 2019). Altogether, 64 individuals were reported, and 12 (19%) exhibit diverse forms of seizures, including focal epilepsy, febrile seizure, nocturnal epilepsy, and epilepsy with multifocal origin. Among those individuals, 11 were heterozygous for a LoF variant, and one was heterozygous for an H1909Y missense variant.

**NAGA** (deleted in 4% of PMS individuals) encodes a lysosomal enzyme involved in the catabolism of glycoproteins, sphingolipids, and oligosaccharides. Deficiency in NAGA results in one of the three types of Schindler disease, a rare and recessive lysosomal storage disorder

characterised by central and peripheral axonopathy associated with rapid psychomotor degradation. This disorder was discovered in two siblings presenting early onset neurodegeneration, seizure disorders, and hypotonia (Desnick and Wang, 1990).

In the case of infantile cerebellar retinal degeneration (ICRD) due to mutations in **ACO2**, the analysis of clinical features of eight individuals from two unrelated and consanguineous families showed that six of them developed generalised or focal epilepsy (Spiegel et al., 2012). All carriers were heterozygous for an S112R missense variant. Recently, a database gathering extensive clinical information on 123 individuals with ACO2 dominant or recessive mutations was developed (https://www.lovd.nl/ACO2). Among 21 patients affected by a recessive neurodegenerative syndrome, 13 (62%) also suffered from seizures (Guehlouz et al., 2021).

While they are listed in the HPO for epilepsy, there is, to our knowledge, no report of epilepsy in carriers of variants affecting *FBLN1* and *TUBGC6P* in the literature.

## 5. The 22q13 region and macro/microcephaly

Microcephaly and macrocephaly are neurological conditions characterised by abnormal cranial circumference. In the case of microcephaly, the head size, measured around the forehead and the occipital protuberance, is above two standard deviations (SD) or more for a given age, ethnicity, and sex (respectively below two SD or more for microcephaly, although in some studies it could be defined below three SD). Interestingly, both of these conditions are reported in patients with PMS (17% with macrocephaly and 16% with microcephaly).

Along the 22q13 region, eight genes are associated with microcephaly according to the annotated HPO terms: **CHKB**, **SBF1**, **TUBGCP6**, **ALG12**, **CYB5R3**, **RRP7A**, **NDUFA6**, **ACO2**; only one gene is associated with macrocephaly: **MLC1**; and two genes to both: **SHANK3** and **TCF20** (Fig. 2).

In a study providing deep phenotyping of 14 individuals with a point mutation in **SHANK3**, one patient displayed microcephaly, and one displayed macrocephaly (De Rubeis et al., 2018). Furthermore, a meta-analysis focused on **SHANK** genes reported an additional case carrying a *de novo* frameshift mutation in **SHANK3** and exhibiting microcephaly (Leblond et al., 2014).

Likewise, different studies have described microcephaly in carriers of homozygous recessive **CHKB** mutations (Mitsuhashi et al., 2011; Haliloglu et al., 2015). Cranial MRI of these microcephalic individuals showed no structural brain abnormalities (Mitsuhashi et al., 2011).

**SBF1** (deleted in 73% of PMS individuals) is a gene coding for an inactive phosphatase and is included in the myotubularin family. **SBF1** is associated with the CMT4B3 subtype of Charcot-Marie-Tooth disease, a disorder of the peripheral nervous system (Nakhro et al., 2013; Gang et al., 2020). This specific subtype is characterised by autosomal recessive inheritance and impacts both the sensory/motor axons and the myelin. Early onset microcephaly often co-occurs with the neuropathy caused by recessive **SBF1** mutations, as observed in siblings of four different families (Flusser et al., 2018).

**TUBGCP6** (deleted in 70% of PMS individuals) is a member of the g-tubulin ring complex family. The encoded protein plays a major role in microtubule nucleation at the centrosome and is, therefore, essential for cell division and cell migration. Interestingly, recessive mutations in **TUBGCP6** have been recurrently detected in patients with microcephaly associated with retinopathy (Puffenberger et al., 2012; Martin et al., 2014; Hull et al., 2019; Shurygina et al., 2020).

Concerning **MLC1**, patients with MLC disease are characterised by progressive macrocephaly (Yalçinkaya et al., 2003; van der Knaap et al., 2012). This phenotype develops during the very early infancy of patients, generally within the first four weeks before a stabilisation of the head growth (van der Knaap et al., 2012).

In the case of **ALG12**, patients with CGC-ALG12 carrying compound heterozygous or homozygous variants often present progressive
microcephaly (Tahata et al., 2019; Hiraide et al., 2021; Nicotera et al., 2021).

Microcephaly has also been observed in a case with RHM2 carrying two heterozygous mutations in *CYB5R3* (Mannino et al., 2018). In addition, among 50 patients with RHM2 due to recessive variants in *CYB5R3*, more than half displayed microcephaly (Nicita et al., 2022).

**RRP7A** (deleted in 6% of PMS individuals) codes for a protein expressed in radial glial cells and ependymal cilia during brain development. Recently, a deleterious RRP7A homozygous missense variant in RRP7A was identified in eight members of a consanguineous family with severe microcephaly (Farooq et al., 2020). Zebrafish homozygous for a frameshift mutation in RRP7A also display smaller brain sizes than wild-type fish (Farooq et al., 2020). Reduction in brain size may result from a defect in rRNA processing and impaired primary cilia resorption leading to alteration of cell proliferation in the course of neurogenesis (Farooq et al., 2020).

Patients with **TCF20** mutations can exhibit either microcephaly or macrocephaly. For macrocephaly, 13 cases heterozygous for **TCF20** LoF variants have been published (Torti et al., 2019; Vetrini et al., 2019). Microcephaly is less reported in patients with **TCF20** mutations. However, a reduction in brain size is observed in homozygous *Tcf20* knockout mice (Feng et al., 2020).

**NDUFA6** (deleted in 4% of PMS individuals) codes for a nuclear subunit of NADH dehydrogenase, representing the first electron entry in the respiratory chain. Clinical evaluation of one carrier of a homozygous pathogenic mutation in *NUDFA6* shows a reduction of head growth noticed at 5 years old (Alston et al., 2018).

Finally, a cohort of 16 patients homozygous or compound heterozygous for **ACO2** mutations showed that 75% of them were diagnosed with severe microcephaly. Head growth defects in these patients occurred between the first and third years of age (Sharkia et al., 2019).

## 6. The 22q13 region and synaptic genes

Synapses play major roles in information processing, and their dysfunction can lead to brain disorders such as neurodevelopmental and psychiatric disorders (Bourgeron, 2009; Lepeta et al., 2016). A large number of synaptic proteins participate in developing and maintaining synapses to ensure their organisation and activities. Cellular localisation and functions of synaptic proteins are well documented in SynGO, a knowledge base initiated by a scientific consortium (https://www.syngo portal.org/). Within the 22q13 region, only four genes are listed in

SynGO: SEPTIN3 and ARFGAP3 are classified as coding for presynaptic proteins, SHANK3 codes for a postsynaptic protein, and PACSIN2 appears as a modulator of the synaptic transmission (Fig. 3).

**SHANK3** is enriched at the postsynaptic density of glutamatergic neurons. Composed of five domains, SHANK3 acts as a master scaffolder by interacting with multiple other synaptic proteins. Partners of SHANK3 comprise glutamatergic metabotropic, NMDA and AMPA receptors, signalling proteins, other intermediate scaffolding proteins, and cytoskeleton proteins (Sheng and Kim, 2000). The role of SHANK3 is to maintain the protein architecture of the synapse. In addition, it participates in receptor signalling and thus is essential for synaptic transmission, plasticity, and homeostasis (Verpelli et al., 2011; Tatavarty et al., 2020). During brain development, SHANK3 is also involved in forming dendritic spines (Durand et al., 2012; Gouder et al., 2019; Peça et al., 2011).

**PACSIN2** (deleted in 10% of PMS individuals) belongs to a protein kinase family important for endocytosis processes (Kessels and Qualmann, 2004). These proteins interact with multiple molecules implicated in different steps of endocytosis, including N-WASP, which activate the Arp2/3 complex and dynamin, with both acting in the plasma membrane fission for vesicle release in the cytosol (Kessels and Qualmann, 2004). PACSIN proteins also recycle AMPA receptors at the plasma membrane required for long-term depression induction, which is a cellular mechanism underlying plasticity, learning, and memory. AMPA receptor trafficking at the postsynaptic membrane is induced by the binding of PACSIN1 and PACSIN2 to proteins involved in the endocytosis of AMPA receptors, such as PICK1 (Anggono et al., 2013).

**ARFGAP3** (deleted in 9% of PMS individuals) proteins are located at the Golgi apparatus in the presynaptic area of neurons. The proteins regulate the retrograde transport of vesicles from the Golgi to the endoplasmic reticulum and within the intra-Golgi cisternae (Spang et al., 2010). ARFGAP3 participates in the COPI coat assembly, a coat protein complex that initiates the budding process on the cis-Golgi membrane. This COPI coat assembly is critical during vesicle formation (Kartberg et al., 2010). ARFGAP3 functions also include the regulation of Arf1 activities and endocytic membrane traffic in retinal photoreceptor synapses (Dembla et al., 2014).

**SEPTIN3** (deleted in 3% of PMS individuals) is a member of the large GTPase protein family and is predominantly expressed at the presynaptic nerve terminal of mature neurons across many different brain regions (Xue et al., 2004; Peterson et al., 2007). Septin proteins associate with each other to form hetero-octamer or hetero-hexamer structures.

<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Synaptic locations and functions of genes of the 22q13 region. SynGO sunburst plot of the four synaptic genes in the 22q13 region (https://www.syngo portal.org/). The first plot shows the compartmental synaptic location, and the second shows the genes' biological function. The inner layers provide general information, and the outer layer provides more specific detail. Sections are coloured if a gene of the query list belongs to the category. Grey sections correspond to categories with no genes in the query list. The 22q13 region contains two genes coding for presynaptic proteins (SEPTIN3; ARFGAP3) and one gene coding for a postsynaptic protein (SHANK3). Biological functions include modulation of chemical synaptic transmission (SHANK3; PACSIN2), structural constituent of postsynaptic density (SHANK3), and presynaptic endocytosis (ARFGAP3).</figcaption></figure>
One such complex comprises SEPTIN2, SEPTIN3, SEPTIN6, and SEP-
TIN7, with SEPTIN3 placed at the centre of the resulting filament
(DeRose et al., 2020). Septin proteins are involved in diverse functions,
including exocytosis, neural cell migration, neuritogenesis, and spino-
genesis (Tokhtaeva et al., 2015; Ageta-Ishihara and Kinoshita, 2021).
However, due to possible redundant functions of these proteins, it is still
challenging to delineate the precise role of SEPTIN3 in the brain.

7. Discussion

The vast majority of the individuals with PMS display global devel-
opmental delay, ID, absent or delayed speech, neonatal hypotonia
together with minor dysmorphic features (long eyelashes, dolicho-
cephaly, bulbous nose, large fleshy hands, and others). They can also
suffer from other non-specific clinical features, including epilepsy,
macrocephaly, macrocephaly, and sleep disorders, as well as complica-
tions related to the kidney, the heart, and the ophthalmic system (Schön
et al., this issue). In most cases, PMS co-occurs with psychiatric condi-
tions such as ASD, depressive episodes, anxiety symptoms, and bipolar
disorder. In addition, regression, or loss of motor skills, is often observed
in adolescence, with possible recovery occurring after a variable period
of time (Kohlenberg et al., 2020; Reierson et al., 2017; van Balkom et al.,
this issue).

In individuals with SHANK3-related PMS, there is also a wide vari-
ability of the type and the size of the deletion. The distribution of the
breakpoint position is mostly uniform along the 22q13 region, with the
exception of a recurrent breakpoint within the SHANK3 gene (Fig. 1B,
Bonaglia et al., 2006; Durand et al., 2007). More than half of individuals
with PMS carrying a 22q13 deletion are haploinsufficient for at least 34
genes, including seven genes previously associated with NDD (Fig. 1C).
Patients with larger deletion are more likely to present macrocephaly,
severe developmental delay, or some dysmorphic features (Soorya et al.,
2013; Sarasua et al., 2014b). In addition, the absence of speech is more
frequent in individuals with a deletion disrupting the genomic region
from position 42.6–46.3 Mb (Tabet et al., 2017). Other genomic regions
could be associated with gastrointestinal disorders, ophthalmic features,
and epilepsy, although there are inconsistent results between studies
(Tabet et al., 2017; Jain et al., 2022).

Due to its terminal position on chromosome 22 and its biological
functions, SHANK3 has been considered the primary gene for PMS
neurological features. Patients with a point mutation in SHANK3 share
the core symptoms and similar frequency with patients carrying a
deletion of the 22q13 region (Schön et al., this issue). However, in-
dividuals with SHANK3-unrelated PMS have overlapping PMS pheno-
types suggesting the contribution of other genes at 22q13 than SHANK3.
For example, PHF21B corresponds to a candidate NDD gene which is
deleted in 13 individuals over the 21 individuals carrying an interstitial
deletion of variable size not affecting SHANK3 (Supplemental Fig. 1).
PHF21B codes for a PHD finger protein specifically expressed in the
cortex and is critical for neural stem cell differentiation during brain
formation (Basu et al., 2020). The PHF21B gene was proposed as a
susceptibility gene for major depression in humans (Wong et al., 2017),
and mice deleted for Phf21B display an impaired social memory in
addition to a reduced expression of several synaptic genes (Chin et al.,
2022).

We reported information on HC-NDD or candidate NDD genes that
could, in addition to SHANK3 haploinsufficiency, contribute to the large
diversity of neurological phenotypes developed by individuals with PMS
(Fig. 1). TCF20, SCO2, and ACO2 are HC-NDD genes with an autosomal
dominant inheritance pattern. Deletions of these genes can therefore
have a significant impact on the PMS phenotype. SCO2 is located in the
subterminal part of chromosome 22 and is frequently deleted in patients
with PMS (74% of the patients presented here). Haploinsufficiency of
SCO2 may increase the risk of developing myopia, although this
phenotype is not often observed in individuals with PMS (Tran-Viet
et al., 2013). Other symptoms associated with Leigh syndrome should be

considered with precaution, as mutations causing this disorder follow a
recessive inheritance pattern. ACO2 and TCF20 are only involved in
larger deletions (9.4 Mb and 8.7 Mb, respectively) which affect fewer
patients (<5% of the patients presented here). In a subset of individuals
with PMS with very large deletions, TCF20 could add to the severity of a
large set of clinical features observed, such as ID, language delay,
autism, hypotonia, seizures, dysmorphic facial features, macrocephaly,
gastrointestinal, and ophthalmologic abnormalities (Torti et al., 2019;
Vetrini et al., 2019). The rare patients with a 22q13 deletion disrupting
ACO2 could be more susceptible to developing gradual optical defects
during their infancy since ocular function degeneration represents the
main phenotype caused by a dominant or recessive mutation in ACO2
(Charif et al., 2021; Guehlouz et al., 2021).

For a large number of HC-NDD or candidate NDD genes located in
the 22q13 region, inheritance follows a recessive model (Fig. 1A). For
some patients, the deletion could unmask a heterozygous variant
affecting these genes on the non-deleted allele located on the intact
chromosome 22 and therefore increasing the risk of developing addi-
tional clinical features. This mechanism has been described in other
deletion syndromes, such as Smith-Magenis syndrome (DeRose et al.,
2020) and the 16p11.2 syndrome (Pebrel-Richard et al., 2014). Such LoF
mutations on the intact chromosome 22 might be more frequent than
expected since many genes with recessive inheritance are relatively
tolerant to LoF mutations (pLI< 0.9; LOEF ≥0.35, Supplemental Fig.
4). TYMP and NDUFA6 are two examples of recessive genes tolerant to
LoF variants and critical for mitochondrial functioning (Alston et al.,
2018; Bax, 2019). Deleterious mutations in the non-deleted allele of
these genes could affect organs with high energy demand, such as the
brain, but also the heart, the skeletal muscle, and the kidney, and cause
cardiac defects and renal disorders observed in patients with PMS.
Another interesting example of such LoF tolerant and recessive gene is
ARSA, which has been suggested to increase the risk of regression in
patients with PMS (De Coo et al., this issue). MLD resulting from the
unmasking of a missense variant was observed in a PMS patient pre-
senting with regression (Ahn et al., 2020). While it seems that there are
no genes subject to genomic imprinting in the 22q13 region, epigenetic
mechanisms might also contribute to the clinical heterogeneity of PMS.
Indeed, patients carrying large deletions (>1 Mb) display a distinct DNA
methylation pattern compared to control individuals and patients car-
rying smaller deletions or point mutations in SHANK3 (Schenkel et al.,
2021). This specific methylation pattern is associated with a similar
metabolic profile (e.g., reduced utilisation of aerobic energy and
abnormal response to metabolic stress). Interestingly, patients with PMS
showing these epigenetic and metabolic patterns are all deleted for
BRD1, a candidate NDD gene encoding a protein interacting with DNA
and histone tails.

We found 13 genes listed as epilepsy genes in HPO (Fig. 2), but weak
evidence from the literature for FBLN1 and TUBGCP6. While CELSR1 is
not part of the HPO epilepsy list, it could be of particular interest as it is
located in the region previously associated with a higher risk of devel-
oping epilepsy (Jain et al., 2022). Moreover, a recent study suggested a
possible association between epilepsy and variants detected in CELSR3,
a paralog of CELSR1 (Li et al., 2022). Interestingly, ARSA listed in the
epilepsy HPO term is associated with morphologic changes of the white
matter that could explain the increased risk of developing seizures in
individuals with deficiency for this gene (De Coo et al., this issue).

For abnormal head growth, we presented 10 genes associated with
microcephaly and 3 genes associated with macrocephaly (Fig. 2), but
other genes not listed in these HPO terms could be considered. For
example, a minimal region shared by all individuals with SHANK3-un-
related PMS highlighted the possible role of PARVB in macrocephaly
(Disciglio et al., 2014).

The 22q13 region contains four synaptic genes: SHANK3, ARFGAP3,
SEPTIN3, and PACSIN2 (Fig. 3). They ensure the correct functioning of
synapse and synaptic transmission. Alterations of synaptic genes are
often associated with neuropsychiatric conditions or NDD. For example,
mutations in *SHANK3* have been reported in multiple neuropsychiatric conditions, including ASD, schizophrenia, and bipolar disorders (Durand et al., 2007; Gauthier et al., 2010; Leblond et al. 2014; de Sena Cortabitarte et al., 2017). *SHANK3* is also strongly associated with ID, which represents a frequent clinical feature of PMS. Higher susceptibility for psychiatric disorders in PMS could also be due to brain-expressed genes such as *MLC1*, which has been associated with catatonic schizophrenia in several studies (Meyer et al., 2001; Verma et al., 2005; Selch et al., 2007). Postsynaptic genes may also include the candidate NDD gene *MAPK8IP2*, which is enriched in the postsynaptic densities in mice (Giza et al., 2010).

We focused this review on genes that could contribute to the neurological phenotypes of individuals with PMS. An overview of genes within the 22q13 region and their possible contribution to other PMS phenotypes, such as gastrointestinal and urinary tract malformations or heart defects, was previously reported (Ricciardello et al., 2021). Additional lists of genes associated with HPO terms described by Schön et al. are available in Supplemental Table 1. Interestingly, most HC-NDD genes are associated with more than 10 HPO terms related to symptoms observed in individuals with PMS.

We also limited this review to the protein-coding genes, but another trail to explore is the role of the non-coding regions, which represent 98% of the genome. Non-coding DNA includes promoter, 3'UTR, 5'UTR, enhancer, silencer, non-coding RNA, and TAD boundaries that regulate gene expression (Turner and Eichler, 2019). The contribution of the non-protein-coding segment of the genome in diseases has been studied in multiple genetic brain disorders (Medico-Salsench et al., 2021). CNVs disrupting non-coding regions located near epilepsy genes were found to be enriched in patients with epilepsy and less frequent in controls (Monlong et al., 2018). Likewise, variants falling in UTR regions, foetal promoters, and embryonic enhancers were significantly enriched in autistic individuals compared to their undiagnosed siblings (Turner et al., 2017). Genome-wide association studies also highlighted the importance of loci associated with a difference in gene expression during the developmental period and allowed the discovery of expression quantitative trait loci (eQTL) that collectively increase the risk of developing neuropsychiatric conditions like bipolar disorder, schizophrenia, and attention deficit with or without hyperactivity (O'Brien et al., 2018). Analysis of eQTL, therefore, represents a promising approach to improve our understanding of phenotypic variability in complex disorders such as PMS.

Finally, the diversity of clinical symptoms may also be modulated by rare or common variants located outside the 22q13 region. Additional CNVs disrupting an NDD gene were identified in 65% of patients with PMS (Tabet et al., 2017). Such multiple hits present in the patient genomes, including CNV, SNV, and indel variants, could increase the phenotypic severity following an additive or a synergic model. In autism, full-scale IQ is lower in individuals accumulating more than one clinically relevant variant in NDD genes (Guo et al., 2019). Likewise, another study highlighted a reduced full-scale IQ in individuals carrying more than two high-impact *de novo* variants affecting LoF intolerant genes (Warrier et al., 2022). Common variants could also modulate the severity of the symptoms. For example, the polygenic score for schizophrenia is higher in carriers of 22q11.2 deletion diagnosed with schizophrenia (Davies et al., 2020). In contrast, additional variants could have a protective effect on some clinical features. More research on these additive risk or resilience factors is highly needed.

In summary, this review of the genes at chromosome 22q13 indicates that several genes might influence the complex and heterogeneous clinical trajectories of individuals with PMS. Loss of a single copy of *SHANK3* recapitulates many of the symptoms of PMS, but further studies should consider the surrounding genes at chromosome 22q13, other genetic variations on the remaining non-deleted allele, and more widely in the genome that might modulate the severity of the symptoms. This has importance for appropriate caregiving and clinical trials. Understanding the genotype-phenotype relationship in PMS will require very

large-scale genetic and phenotypic data across countries and the possibility of securely sharing the data with the scientific/medical community and family associations.

## Disclosure of conflict of interest

The authors declare that they have no conflicts of interest related to the publication of this study.

## CRediT authorship contribution statement

**Aline Vitrac:** Writing – original draft, Investigation, Formal analysis, Visualization. **Claire S. Leblond:** Methodology, Formal analysis, Writing – review & editing. **Thomas Rolland:** Methodology, Formal analysis, Writing – review & editing. **Freddy Cliquet:** Methodology, Formal analysis. **Alexandre Mathieu:** Data curation. **Anna Maruani:** Investigation, Resources. **Richard Delorme:** Investigation, Resources. **Michael Schön:** Writing – review & editing. **Andreas M. Grabrucker:** Conceptualization, Writing – review & editing. **Conny van Ravenswaaij-Arts:** Writing – review & editing. **Katy Phelan:** Writing – review & editing. **Anne-Claude Tabet:** Investigation, Resources. **Thomas Bourgeron:** Conceptualization, Writing – original draft, Supervision.

## Data availability

Data will be made available on request.

## Acknowledgements and fundings

We want to thank all families concerned by Phelan-McDermid syndrome and the Phelan-McDermid Foundation for collecting, storing, and sharing the data used in this review. This study has been supported by the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability (ERN-ITHACA). ERN-ITHACA is partly co-funded by the Health Programme of the European Union. Funding was also obtained from the European Union's Horizon 2020 research and innovation programme under the EJP RD COFUND-EJP N° 825575. This work was also funded by Institut Pasteur, the Bettencourt-Schueller Foundation, Université de Paris Cité, the Eranet-Neuron (ALTRUISM), the Association Française du Syndrome Phelan-mcDermid and the Association Téhani et les enfants Phelan-McDermid, the GenMed Labex, AIMS-2-TRIALS which received support from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement N° 777394 and the INCEPTION program (Investissement d'Avenir grant ANR-16-CONV-0005). This project has received funding from the European Union's Horizon 2020 research and innovative program CANDY under grant agreement N° 847818. The views expressed here are the responsibility of the author(s) only. The EU Commission takes no responsibility for any use made of the information set out.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmg.2023.104732.

## References

* Ageta-Ishihara, N., Kinoshita, M., 2021. Developmental and postdevelopmental roles of septins in the brain. Neurosci. Res. 7. https://doi.org/10.1016/j.neures.2020.08.006.

* Ahn, H., Seo, G.H., Keum, C., Heo, S.H., Kim, T., Choi, J., et al., 2020. Diagnosis of metachromatic leukodystrophy in a patient with regression and Phelan-McDermid syndrome. Brain Dev. 42, 414–417. https://doi.org/10.1016/j.braindev.2020.02.003.

* Alston, C.L., Heidler, J., Dibley, M.G., Kremer, L.S., Taylor, L.S., Fratter, C., et al., 2018. Bi-Allelic mutations in NDUFA6 establish its role in early-onset isolated
mitochondrial complex I deficiency. Am. J. Hum. Genet. 103, 592-601. https://doi.org/10.1016/j.ajhg.2018.08.013.

Anggono, V., Koç-Schmitz, Y., Widagdo, J., Kormann, J., Quan, A., Chen, C.-M., et al., 2013. PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression. Proc. Natl. Acad. Sci. USA 110, 13976-13981. https://doi.org/10.1073/pnas.1312467110.

Babbs, C., Lloyd, D., Pagnamenta, A.T., Twigg, S.R.F., Green, J., McGowan, S.J., et al., 2014. *De novo* and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder. J. Med. Genet. 51, 737-747. https://doi.org/10.1136/jmedgenet-2014-102582.

Bakare, A.B., Lesnefsky, E.J., Iyer, S., 2021. Leigh syndrome: a tale of two genomes. Front. Physiol. 12, 693734. https://doi.org/10.3389/fphys.2021.693734.

Barboura, I., Reguez, S., Soyah, N., sahli, C.A., Chkioua, L., 2022. The Missense Mutation in ARSA Gene Causes the Juvenile Form of MLD. Clin Genet 75, 57-64. https://doi.org/10.21203/rs.3.rs-1374810/v1.

Basu, A., Mestres, I., Sahu, S.K., Tiwari, N., Khongwir, B., Baumgart, J., et al., 2020. Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation. Genes Dev. 34, 1190-1209. https://doi.org/10.1101/gad.333906.119.

Bax, B.E., 2019. Mitochondrial neurogastrointestinal encephalomyopathy: approaches to diagnosis and treatment. J. Transl. Genet. Genomics. https://doi.org/10.20517/jtg.2020.08.

Boeckers, T.M., Bockmann, J., Kreutz, M.R., Gundelfinger, E.D., 2002. ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease: role of ProSAP/Shank in PSD organization. J. Neurochem. 81, 903-910. https://doi.org/10.1046/j.1471-4159.2002.00931.x.

Bonaglia, M.C., Bertuzzo, S., Ciaschini, A.M., Discepoli, G., Castiglia, L., Romaniello, R., et al., 2020. Targeted next-generation sequencing identifies the disruption of the SHANK3 and RYR2 genes in a patient carrying a de novo t(1;22)(q43;q13.3) associated with signs of Phelan-McDermid syndrome. Mol. Cytogenet. 13, 22. https://doi.org/10.1186/s13039-020-00490-6.

Bonaglia, M.C., Giorda, R., Beri, S., De Agostini, C., Novara, F., Fichera, M., et al., 2011. Molecular mechanisms generating and stabilizing terminal 22q13 deletion in 44 subjects with phelan/McDermid syndrome. PLoS Genet. 7, e1002173 https://doi.org/10.1371/journal.pgen.1002173.

Bonaglia, M.C., Giorda, R., Mani, E., Aceti, G., Anderlid, B.-M., Baroncini, A., et al., 2006. Identification of a recurrent breakpoint within the SHANK3 gene in the 22q13.3 deletion syndrome. J. Med. Genet. 43, 822-828. https://doi.org/10.1136/jmg.2005.038604.

Bourgeron, T., 2009. A synaptic trek to autism. Curr. Opin. Neurobiol. 4. https://doi.org/10.1016/j.conlb.2009.06.003.

Charif, M., Gueguen, N., Ferré, M., Elkharat, Z., Khiati, S., LeMao, M., et al., 2021. Dominant ACO2 mutations are a frequent cause of isolated optic atrophy. Brain Commun. 3, fcbab063. https://doi.org/10.1093/braincomms/fcbab063.

Chin, E.W.M., Ma, Q., Ruan, H., Chin, C., Somasundaram, A., Zhang, C., et al., 2022. The epigenetic reader PHF21B modulates murine social memory and synaptic plasticity-related genes. JCI Insight 7, e158081. https://doi.org/10.1172/jci insight.158081.

Davies, R.W., Fiksinski, A.M., Breetvelt, E.J., Williams, N.M., Hooper, S.R., Monfeuga, T., et al., 2020. Using common genetic variation to examine phenotypic expression and risk prediction in 22q11.2 deletion syndrome. Nat. Med. 26, 1912-1918. https://doi.org/10.1038/s41591-020-1103-1.

De Coo, I., Jesse, S., Le, T.-L., Sala, C., European Phelan-McDermid Syndrome Consortium, Bourgeron, T, (this issue). Consensus recommendations on Epilepsy in Phelan-McDermid syndrome.

De Rubeis, S., Siper, P.M., Durkin, A., Weissman, J., Muratet, F., Halpern, D., et al., 2018. Delineation of the genetic and clinical spectrum of Phelan-McDermid syndrome caused by SHANK3 point mutations. Mol. Autism. 9, 31. https://doi.org/10.1186/s13229-018-0205-9.

de Sena Cortartarte, A., Degenhardt, F., Strohmaier, J., Lang, M., Weiss, B., Roeth, R., et al., 2017. Investigation of SHANK3 in schizophrenia. Am. J. Med. B Neuropsychiatr. Genet. 174, 390-398. https://doi.org/10.1002/ajmg.b.32528.

Delling, J.P., Boeckers, T.M., 2021. Comparison of SHANK3 deficiency in animal models: phenotypes, treatment strategies, and translational implications. J. Neurodev. Disord. 13, 55. https://doi.org/10.1186/s11689-021-09397-8.

Dembla, M., Wahl, S., Katiyar, R., Schmitz, F., 2014. ArfGAP3 is a component of the photoreceptor synaptic ribbon complex and forms an NAD(H)-Regulated, redox-sensitive complex with RIBEYE that is important for endocytosis. J. Neurosci. 34, 5245-5260. https://doi.org/10.1523/JNEUROSCI.3837-13.2014.

Denayer, A., Van Esch, H., de Ravel, T., Frijns, J.-P., Van Buggenhout, G., Vogels, A., et al., 2012. Neuropsychopathology in 7 patients with the 22q13 deletion syndrome: presence of bipolar disorder and progressive loss of skills. Mol. Syndromol. 3, 14-20. https://doi.org/10.1159/000339119.

DeRose, B.T., Kelley, R.S., Ravi, R., Kokona, B., Beld, J., Spiliotis, E.T., et al., 2020. Production and analysis of a mammalian septin hetero-octamer complex. Cytoskeleton 77, 485-499. https://doi.org/10.1002/cm.21643.

Desnick, R.J., Wang, A.M., 1990. Schindler Disease: An Inherited Neuroaxonal Dystrophy Due to -N-Acetylgalactosaminidase Deficiency. J Inherit Metab Dis. 11. https://doi.org/10.1007/BF01799512.

Disciglio, V., Rizzo, C.L., Mencarelli, M.A., Mucciolo, M., Marozza, A., Di Marco, C., et al., 2014. Interstitial 22q13 deletions not involving SHANK3 gene: a new contiguous gene syndrome. Am. J. Med. Genet. A. 164, 1666-1676. https://doi.org/10.1002/ajmg.a.36513.

Dubey, M., Brouwers, E., Hamilton, E.M.C., Stiedl, O., Bugiani, M., Koch, H., et al., 2018. Seizures and disturbed brain potassium dynamics in the leukodystrophy

megalencephalic leukoencephalopathy with subcortical cysts. Ann. Neurol. 83, 636-649. https://doi.org/10.1002/ana.25190.

Durand, C.M., Betancur, C., Boeckers, T.M., Bockmann, J., Chaste, P., Fauchereau, F., et al., 2007. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat. Genet. 39, 25-27. https://doi.org/10.1038/ng1933.

Durand, C.M., Perroy, J., Loll, F., Perrais, D., Fagni, L., Bourgeron, T., et al., 2012. SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism. Mol. Psychiatr. 17, 71-84. https://doi.org/10.1038/mp.2011-57.

Farooq, M., Lindbæk, L., Krogh, N., Doganli, C., Keller, C., Mönnich, M., et al., 2020. RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis. Nat. Commun. 11, 5816. https://doi.org/10.1038/s41467-020-19658-0.

Feng, C., Zhao, J., Ji, F., Su, L., Chen, Y., Jiao, J., 2020. TCF 20 dysfunction leads to cortical neurogenesis defects and autistic-like behaviors in mice. EMBO Rep. 21 https://doi.org/10.15252/embr.201949239.

Flusser, H., Halperin, D., Kadir, R., Shorer, Z., Shelef, I., Birk, O.S., 2018. Novel SBF1 splice-site null mutation broadens the clinical spectrum of Charcot-Marie-Tooth type 4B3 disease. Clin. Genet. 94, 473-479. https://doi.org/10.1111/cge.13419.

Gang, Q., Bettencourt, C., Holton, J., Lovejoy, C., Chelban, V., Oconnor, E., et al., 2020. A novel frameshift deletion in autosomal recessive SBF1-related syndromic neuropathy with necklace fibres. J. Neurol. 267, 2705-2712. https://doi.org/10.1007/s00415-020-09827-y.

Gauthier, J., Champagne, N., Lafrenière, R.G., Xiong, L., Spiegelman, B., Drustein, E., et al., 2010. De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia. Proc. Natl. Acad. Sci. USA 107, 7863-7868. https://doi.org/10.1073/pnas.0906232107.

Giza, J., Urbanski, M.J., Prestori, F., Bandyopadhyay, B., Yam, A., Friedrich, V., et al., 2010. Behavioral and cerebellar transmission deficits in mice lacking the autism-linked gene islet brain-2. J. Neurosci. 30, 14805-14816. https://doi.org/10.1523/JNEUROSCI.1161-10-2010.

Gouder, L., Vitrac, A., Goubran-Botros, H., Danckaert, A., Tinevez, J.-Y., André-Leroux, G., et al., 2019. Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations. Sci. Rep. 9, 94. https://doi.org/10.1038/s41598-018-36993-x.

Gu, Z., Gu, L., Eils, R., Schlesner, M., Brors, B., 2014. Circlize implements and enhances circular visualization in R. Bioinformatics 30, 2811-2812. https://doi.org/10.1093/bioinformatics/btu393.

Guehlouz, K., Foulonneau, T., Amati-Bonneau, P., Charif, M., Colin, E., Bris, C., et al., 2021. ACO2 clinicobiological dataset with extensive phenotype ontology annotation. Sci. Data 8, 205. https://doi.org/10.1038/s41597-021-00984-x.

Guo, H., Duyzend, M.H., Coe, B.P., Baker, C., Hoekzema, K., Gerdts, J., et al., 2019. Genome sequencing identifies multiple deleterious variants in autism patients with more severe phenotypes. Genet. Med. 21, 1611-1620. https://doi.org/10.1038/s41436-018-0380-2.

Haeuptle, M.A., Hennet, T., 2009. Congenital disorders of glycosylation: an update on defects affecting the biosynthesis of dolichol-linked oligosaccharides. Hum. Mutat. 30, 1628-1641. https://doi.org/10.1002/humu.21126.

Haliloglu, G., Talim, B., Sel, C.G., Topaloglu, H., 2015. Clinical characteristics of megaclonal congenital muscular dystrophy due to choline kinase beta gene defects in a series of 15 patients. J. Inherit. Metab. Dis. 38, 1099-1108. https://doi.org/10.1007/s10545-015-9856-2.

Han, J., Holder Jr., J.L., Schaaf, C.P., Lu, H., Chen, H., Kang, H., et al., 2013. SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties. Nature 503, 72-77. https://doi.org/10.1038/nature12630.

Hiraide, T., Wada, Y., Matsubayashi, T., Kadoya, M., Masunaga, Y., Ohkubo, Y., et al., 2021. Novel ALG12 variants and hydronephrosis in siblings with impaired N-glycosylation. Brain Dev. 43, 945-951. https://doi.org/10.1016/j.braindev.2021.05.013.

Holder, J.L., Quach, M.M., 2016. The spectrum of epilepsy and electroencephalographic abnormalities due to SHANK3 loss-of-function mutations. Epilepsia 57, 1651-1659. https://doi.org/10.1111/epi.13506.

Hull, S., Arno, G., Ostergaard, P., Pontikos, N., Robson, A.G., Webster, A.R., et al., 2019. Clinical and molecular characterization of familial exudative vitreoretinopathy associated with microcephaly. Am. J. Ophthalmol. 207, 87-98. https://doi.org/10.1016/j.ajo.2019.05.001.

Jain, L., Oberman, L.M., Beamer, L., Cascio, L., May, M., Srikanth, S., et al., 2022. Genetic and metabolic profiling of individuals with Phelan-McDermid syndrome presenting with seizures. Clin. Genet. 101, 87-100. https://doi.org/10.1111/cge.14074.

Jaksch, M., Ogilvie, I., Yao, J., Kortenhaus, G., Bresser, H.-G., Gerbitz, K.-D., et al., 2000. Mutations in SCO2 are associated with a distinct form of hypertrophic cardiomyopathy and cytochrome c oxidase deficiency. Hum. Mol. Genet. 9, 795-801. https://doi.org/10.1093/hmg/9.5.795.

Jin, C., Zhang, Y., Kim, S., Kim, Y., Lee, Y., Han, K., 2018. Spontaneous seizure and partial lethality of juvenile Shank3-overexpressing mice in C57BL/6 J background. Mol. Brain 11, 57. https://doi.org/10.1186/s13041-018-0403-6.

Kang, D.-H., Lee, S.D., Hong, Y.-H., Lee, S.-T., Jeon, B.R., Lee, Y.K., et al., 2010. Identification of a novel splicing mutation in the ARSA gene in a patient with late-infantile form of metachromatic leukodystrophy. Ann. Lab. Med. 30, 516-520. https://doi.org/10.3343/jlkm.2010.30.5.516.

Karczewski, K.J., Francioli, L.C., Tiao, G., Cummings, B.B., Alfordi, J., Wang, Q., et al., 2020. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581, 434-443. https://doi.org/10.1038/s41586-020-2308-7.
Kartberg, F., Asp, L., Dejgaard, S.Y., Smedh, M., Fernandez-Rodriguez, J., Nilsson, T., et al., 2010. ARFGAP2 and ARFGAP3 are essential for COPI coat assembly on the Golgi membrane of living cells. J. Biol. Chem. 285, 36709-36720. https://doi.org/10.1074/jbc.M110.180380.

Kessels, M.M., Qualmann, B., 2004. The syndapin protein family: linking membrane trafficking with the cytoskeleton. J. Cell Sci. 117, 3077-3086. https://doi.org/10.1242/jcs.01290.

Kohlenberg, T.M., Trelles, M.P., McLarney, B., Betancur, C., Thurm, A., Kolevzon, A., 2020. Psychiatric illness and regression in individuals with Phelan-McDermid syndrome. J. Neurodev. Disord. 12, 7. https://doi.org/10.1186/s11689-020-9309-6.

Koza, S., Tabet, A. -C., Bonaglia, C., Andres, S., Anderlid, B. M., Aten, E., et al. (this issue) Consensus Recommendations on Counseling in Phelan-McDermid Syndrome; with Special Attention to Recurrence Risk and to Ring Chromosome 22.

Kryuchkova-Mostacci, N., Robinson-Rechavi, M., 2016. A benchmark of gene expression tissue-specificity metrics. Briefings Bioinf. https://doi.org/10.1093/bib/bbw008 bw008.

Lake, N.J., Compton, A.G., Rahman, S., Thorburn, D.R., 2016. Leigh syndrome: one disorder, more than 75 monogenic causes: Leigh Syndrome. Ann. Neurol. 79, 190-203. https://doi.org/10.1002/ana.24551.

Leblond, C.S., Le, T.-L., Malesys, S., Cliquet, F., Tabet, A.-C., Delorme, R., et al., 2021. Operative list of genes associated with autism and neurodevelopmental disorders based on database review. Mol. Cell. Neurosci. 113, 103623 https://doi.org/10.1016/j.mcn.2021.103623.

Leblond, C.S., Nava, C., Polge, A., Gauthier, J., Huguet, G., Lumbroso, S., et al., 2014. Meta-analysis of SHANK mutations in autism spectrum disorders: a gradient of severity in cognitive impairments. PLoS Genet. 10, e1004580 https://doi.org/10.1371/journal.pgen.1004580.

Leegwater, P.A.J., Yuan, B.Q., van der Steen, J., Mulders, J., Könst, A.A.M., Boor, P.K.I., et al., 2001. Mutations of MLCI (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts. Am. J. Hum. Genet. 68, 831-838. https://doi.org/10.1086/319519.

Lelieveld, S.H., Reijnders, M.R., Pfundt, R., Yntema, H.G., Kamsteeg, E.-J., de Vries, P., et al., 2016. Meta-analysis of 2,104 trios provides support for 10 new genes for intellectual disability. Nat Neurosci 19, 1194-1196. https://doi.org/10.1038/nn.4352.

Lepeta, K., Lourenco, M.V., Schweitzer, B.C., Martino Adami, P.V., Banerjee, P., Catuara-Solarz, S., et al., 2016. Synaptopathies: synaptic dysfunction in neurological disorders - a review from students to students. J. Neurochem. 21. https://doi.org/10.1111/jnc.13713.

Lévy, J., Cogan, G., Maruani, A., Maillard, A., Dupont, C., Drunat, S., et al., 2022. Rare and de novo duplications containing TCF20 are associated with a neurodevelopmental disorder. Clin. Genet. 101, 364-370. https://doi.org/10.1111/cge.14099.

Li, J., Lin, S., Qiao, J., Liu, X., Wang, J., Jiang, M., et al., 2022. *CELSR3* variants are associated with febrile seizures and epilepsy with antecedent febrile seizures. CNS Neurosci. Ther. 28, 382-389. https://doi.org/10.1111/cns.13781.

Li, S., Xi, K., Liu, T., Zhang, Y., Zhang, M., Zeng, L., et al., 2020. Fraternal twins with Phelan-McDermid syndrome not involving the SHANK3 gene: case report and literature review. BMC Med. Genom. 13, 146. https://doi.org/10.1186/s12920-020-00802-0.

Mannino, E.A., Pluim, T., Wessler, J., Cho, M.T., Juusola, J., Schrier Vergano, S.A., 2018. Congenital methemoglobinemia type II in a 5-year-old boy. Clin. Case Rep. 6, 170-178. https://doi.org/10.1002/ccr3.1310.

Marelli, C., Hamel, C., Quiles, M., Carlander, B., Larrieu, L., Delettem, C., et al., 2018. ACO2 mutations: a novel phenotype associating severe optic atrophy and spastic paraplegia. Neurol. Genet. 4, e225. https://doi.org/10.1212/NXG.0000000000000000.

Martin, C.-A., Ahmad, I., Klingseisen, A., Hussain, M.S., Bicknell, L.S., Leitch, A., et al., 2014. Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy. Nat. Genet. 46, 12. doi:10.1038/ng.3122.

McFarland, K.N., Liu, J., Landrian, I., Zeng, D., Raskin, S., Moscovich, M., et al., 2014. Repeat interruptions in spinocerebellar ataxia type 10 expansions are strongly associated with epileptic seizures. Neurogenetics 15, 59-64. https://doi.org/10.1007/s10048-013-0385-6.

Medico-Salsench, E., Karkala, F., Lanko, K., Barakat, T.S., 2021. The non-coding genome in genetic brain disorders: new targets for therapy? Essays Biochem. 65, 671-683. https://doi.org/10.1042/EBC20200121.

Medotiev, M.D., Gerber, S., Hubert, L., Delahodde, A., Chretien, D., Gérard, X., et al., 2014. Mutations in the tricarboxylic acid cycle enzyme, aconitase 2, cause either isolated or syndromic optic neuropathy with encephalopathy and cerebellar atrophy. J. Med. Genet. 51, 834-838. https://doi.org/10.1136/jmedgenet-2014-102532.

Meyer, J., Huberth, A., Ortega, G., Syagailo, Y.V., Jatzke, S., Mössner, R., et al., 2001. A missense mutation in a novel gene encoding a putative cation channel is associated with catatonic schizophrenia in a large pedigree. Mol. Psychiatr. 6, 302-306. https://doi.org/10.1038/sj.mp.4000869.

Mitsuhashi, S., Ohkuma, A., Talim, B., Karahashi, M., Koumura, T., Aoyama, C., et al., 2011. A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis. Am. J. Hum. Genet. 88, 845-851. https://doi.org/10.1016/j.ajhg.2011.05.010.

Monlong, J., Girard, S.L., Meloche, C., Cadieus-Dion, M., Andrade, D.M., Lafreniere, R. G., et al., 2018. Global characterization of copy number variants in epilepsy patients from whole genome sequencing. PLoS Genet. 14, e1007285 https://doi.org/10.1371/journal.pgen.1007285.

Musto, E., Gardella, E., Møller, R.S., 2020. Recent advances in treatment of epilepsy-related sodium channelopathies. Eur. J. Paediatr. Neurol. 24, 123-128. https://doi.org/10.1016/j.ejpn.2019.12.009.

Nakhro, K., Park, J.-M., Hong, Y.B., Park, J.H., Nam, S.H., Yoon, B.R., et al., 2013. SET Binding Factor 1 (SBF1) Mutation Causes Charcot-Marie-Tooth Disease Type 4B3. Neurology 19, 1194-1196. https://doi.org/10.1212/WNL.0b013e31829a3421.

Nevado, J., García-Minaúr, S., Palomares-Bralo, M., Vallespín, E., Guillén-Navarro, E., Rosell, J., et al., 2022. Variability in phelan-McDermid syndrome in a cohort of 210 individuals. Front. Genet. 13, 652454 https://doi.org/10.3389/fgenet.2022.652454.

Nicita, F., Sabatini, L., Alesi, V., Lucignani, G., Sallicandro, E., Serra, A., et al., 2022. Neurological and neuroimaging features of CYB5R3-related recessive hereditary methemoglobinemia type II. Brain Sci. 12, 182. https://doi.org/10.3390/brainsci12020182.

Nicotera, A.G., Spoto, G., Cali, F., Romeo, G., Musumeci, A., Vinci, M., et al., 2021. A novel homozygous ALG12 mutation in a patient with CDG type ig: new report of a case with a mild phenotype. Mol. Syndromol. 12, 327-332. https://doi.org/10.1159/000516606.

O'Brien, H.E., Hannon, E., Hill, M.J., Toste, C.C., Robertson, M.J., Morgan, J.E., et al., 2018. Expression quantitative trait loci in the developing human brain and their enrichment in neuropsychiatric disorders. Genome Biol. 19, 194. https://doi.org/10.1186/s13059-018-1567-1.

Palumbo, P., Accadia, M., Leone, M.P., Palladino, T., Stallone, R., Carella, M., et al., 2018. Clinical and molecular characterization of an emerging chromosome 22q13.31 microdeletion syndrome. Am. J. Med. Genet. A. 176, 391-398. https://doi.org/10.1002/ajmg.a.38559.

Pebrel-Richard, C., Debost-Legrand, A., Eymard-Pierre, G., Greze, V., Kemeny, S., Gay-Bellile, M., et al., 2014. An unusual clinical severity of 16p11.2 deletion syndrome caused by unmasked recessive mutation of CLN3. Eur. J. Hum. Genet. 22, 369-373. https://doi.org/10.1038/ehjg.2013.141.

Peça, J., Feliciano, C., Ting, J.T., Wang, W., Wells, M.F., Venkatraman, T.N., et al., 2011. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437-442. https://doi.org/10.1038/nature09965.

Peterson, E.A., Kalikin, L.M., Steels, J.D., Estey, M.P., Trimble, W.S., Petty, E.M., 2007. Characterization of a SEPT9 interacting protein, SEPT14, a novel testis-specific septin. Mamm. Genome 18, 796-807. https://doi.org/10.1007/s00335-007-9065-x.

Phelan, K., Boccuto, L., Powell, C.M., Boeckers, T.M., van Ravenswaaij-Arts, C., Rogers, R.C., et al., 2022. Phelan-McDermid syndrome: a classification system after 30 years of experience. Orphanet J. Rare Dis. 17, 27. https://doi.org/10.1186/s13023-020180-5.

Phelan, K., McDermid, H.E., 2011. The 22q13.3 deletion syndrome (phelan-McDermid syndrome). Mol. Syndromol. 2, 186-201. https://doi.org/10.1159/000334260.

Puffenberger, E.G., Jinks, R.N., Sougnez, C., Cibulskis, K., Willert, R.A., Achilly, N.P., et al., 2012. Genetic mapping and exome sequencing identify variants associated with five novel diseases. PLoS One 7, 15. https://doi.org/10.1371/journal.pone.0028936.

Rasmussen, A., Matsuura, T., Ruano, L., Yescas, P., Ochoa, A., Ashizawa, T., et al., 2001. Clinical and genetic analysis of 4 Mexican families with spinocerebellar ataxia type 10. Ann. Neurol. 50, 234-239. https://doi.org/10.1002/ana.1081.

Reierson, G., Bernstein, J., Froehlich-Santino, W., Urban, A., Purmann, C., Berquist, S., et al., 2017. Characterizing regression in Phelan McDermid Syndrome (22q13 deletion syndrome). J. Psychiatr. Res. 91, 139-144. https://doi.org/10.1016/j.jpsychres.2017.03.010.

Ricciardello, A., Tomaiuolo, P., Persico, A.M., 2021. Genotype-phenotype correlation in Phelan-McDermid syndrome: a comprehensive review of chromosome 22q13 deleted genes. Am. J. Med. Genet. A. 185, 2211-2233. https://doi.org/10.1002/ajmg.a.62222.

Samogy-Costa, C.I., Varella-Branco, E., Monfardini, F., Ferraz, H., Fock, R.A., Barbosa, R.A., et al., 2019. A Brazilian cohort of individuals with Phelan-McDermid syndrome: genotype-phenotype correlation and identification of an atypical case. J. Neurodev. Disord. 11, 13. https://doi.org/10.1186/s11689-019-9273-1.

Sarasua, S.M., Boccuto, L., Sharp, J.L., Dwivedi, A., Chen, C.-F., Rollins, J.D., et al., 2014a. Clinical and genomic evaluation of 201 patients with Phelan-McDermid syndrome. Hum. Genet. 133, 847-859. https://doi.org/10.1007/s00439-014-1423-7.

Sarasua, S.M., Dwivedi, A., Boccuto, L., Chen, C.-F., Sharp, J.L., Rollins, J.D., et al., 2014b. 22q13.2q13.32 genomic regions associated with severity of speech delay, developmental delay, and physical features in Phelan-McDermid syndrome. Genet. Med. 16, 318-328. https://doi.org/10.1038/gim.2013.144.

Schäfgen, J., Cremer, K., Becker, J., Wieland, T., Zink, A.M., Kim, S., et al., 2016. De novo nonsense and frameshift variants of TCF20 in individuals with intellectual disability and postnatal overgrowth. Eur. J. Hum. Genet. 24, 1739-1745. https://doi.org/10.1038/ehjg.2016.90.

Schenkel, L.C., Aref-Eshghi, E., Rooney, K., Kerkhof, J., Levy, M.A., McConkey, H., et al., 2021. DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome. Clin. Epigenet. 13, 2. https://doi.org/10.1186/s13148-020-00990-7.

Schön, M., Lapunzina, P., Nevado, J., Matina, T., Gunnarson, C., Hadziev, K., et al. (this issue), Definition and Clinical Variability of SHANK3-Related Phelan-McDermid Syndrome.

Selch, S., Strobel, A., Haderlein, J., Meyer, J., Jacob, C.P., Schmitt, A., et al., 2007. MLCI polymorphisms are specifically associated with periodic catatonia, a subgroup of chronic schizophrenia. Biol. Psychiatr. 61, 1211-1214. https://doi.org/10.1016/j.biopsych.2006.08.030.

Shaimardanova, A.A., Chulpanova, D.S., Solovyeva, V.V., Mullagulova, A.I., Kitaeva, K. V., Allegrucci, C., et al., 2020. Metachromatic leukodystrophy: diagnosis, modeling,
and treatment approaches. Front. Med. 7, 576221 https://doi.org/10.3389/fmed.2020.576221.

Sharkia, R., Wierenga, K.J., Kessel, A., Azem, A., Bertini, E., Carrozzo, R., et al., 2019. Clinical, radiological, and genetic characteristics of 16 patients with ACO2 gene defects: delineation of an emerging neurometabolic syndrome. J. Inherit. Metab. Dis. 42, 264–275. https://doi.org/10.1002/jimd.12022.

Sheng, M., Kim, E., 2000. The Shank Family of Scaffold Proteins. J Cell Sci 6. https://doi.org/10.1242/jcs.113.11.1851.

Shurygina, M.F., Simonett, J.M., Parker, M.A., Mitchell, A., Grigorian, F., Lifton, J., et al., 2020. Genotype phenotype correlation and variability in microcephaly associated with chorioretinopathy or familial exudative vitreoretinopathy. Investig. Opthalmology Vis. Sci. 61, 2. https://doi.org/10.1167/iovs.61.13.2.

Simenson, K., Oiglane-Shlik, E., Teek, R., Kuuse, K., Ounap, K., 2014. A patient with the classic features of Phelan-McDermid syndrome and a high immunoglobulin E level caused by a cryptic interstitial 0.72-Mb deletion in the 22q13.2 region. Am. J. Med. Genet. A. 164, 806–809. https://doi.org/10.1002/ajmg.a.36358.

Soorya, L., Kolevzon, A., Zweifach, J., Lim, T., Dobry, Y., Schwartz, L., et al., 2013. Prospective investigation of autism and genotype-phenotype correlations in 22q13 deletion syndrome and SHANK3 deficiency. Mol. Autism. 4, 18. https://doi.org/10.1186/2040-2392-4-18.

Spang, A., Shiba, Y., Randazzo, P.A., 2010. Arf GAPS: gatekeepers of vesicle generation. FEBS Lett. 584, 2646–2651. https://doi.org/10.1016/j.febulet.2010.04.005.

Spiegel, R., Pines, O., Ta-Shma, A., Burak, E., Shaag, A., Halvardson, J., et al., 2012. Infantile cerebellar-retinal degeneration associated with a mutation in mitochondrial aconitase, ACO2. Am. J. Hum. Genet. 90, 518–523. https://doi.org/10.1016/j.ajhg.2012.01.009.

Tabet, A.-C., Rolland, T., Ducloy, M., Lévy, J., Buratti, J., Mathieu, A., et al., 2017. A framework to identify contributing genes in patients with Phelan-McDermid syndrome. Npj Genomic. Med. 2, 32. https://doi.org/10.1038/s41525-017-0035-2.

Tahata, S., Gunderson, L., Lanpher, B., Morava, E., 2019. Complex phenotypes in ALG12-congenital disorder of glycosylation (ALG12-CDG): case series and review of the literature. Mol. Genet. Metab. 128, 409–414. https://doi.org/10.1016/j.ymgme.2019.08.007.

Tatavarty, V., Torrado Pacheco, A., Groves Kuhnle, C., Lin, H., Koundinya, P., Miska, N. J., et al., 2020. Autism-associated Shank3 is essential for homeostatic compensation in rodent V1. Neuron 106, 769–777.e4. https://doi.org/10.1016/j.neuron.2020.02.033.

Tay, S.K.H., Shanske, S., Kaplan, P., DiMauro, S., 2004. Association of mutations in SCO2, a cytochrome c oxidase assembly gene, with early fetal lethality. Arch. Neurol. 61, 950. https://doi.org/10.1001/archneur.61.6.950.

Tokhtaeva, E., Capri, J., Marcus, E.A., Whitelegge, J.P., Khuzakhmetova, V., Bukharaeva, E., et al., 2015. Septin dynamics are essential for exocytosis. J. Biol. Chem. 290, 18. https://doi.org/10.1074/jbc.M114.616201.

Torti, E., Keren, B., Palmer, E.E., Zhu, Z., Afenjar, A., Anderson, Ilse J., et al., 2019. Variants in TCF20 in neurodevelopmental disability: description of 27 new patients and review of literature. Genet. Med. 21, 2036–2042. https://doi.org/10.1038/s41436-019-0454-9.

Tozawa, T., Nishimura, A., Ueno, T., Shikata, A., Taura, Y., Yoshida, T., et al., 2021. Complex hereditary spastic paraplegia associated with episodic visual loss caused by ACO2 variants. Hum. Genome Var. 8, 4. https://doi.org/10.1038/s41439-021-00136-y.

Tran-Viet, K.-N., Powell, C., Barathi, V.A., Klemm, T., Maurer-Stroh, S., Limviphuvadh, V., et al., 2013. Mutations in SCO2 are associated with autosomal-

dominant high-grade myopia. Am. J. Hum. Genet. 92, 820–826. https://doi.org/10.1016/j.ajhg.2013.04.005.

Turner, T.N., Coe, B.P., Dickel, D.E., Hoekzema, K., Nelson, B.J., Zody, M.C., et al., 2017. Genomic patterns of de novo mutation in simplex autism. Cell 171, 710–722.e12. https://doi.org/10.1016/j.cell.2017.08.047.

Turner, T.N., Eichler, E.E., 2019. The role of de novo noncoding regulatory mutations in neurodevelopmental disorders. Trends Neurosci. 42, 115–127. https://doi.org/10.1016/j.tins.2018.11.002.

van Balkom, I.D.C., Burdeus-Olavarrieta, M., Cooke, J., de Cuba, A.G., Turner, A., The European Phelan-McDermid Syndrome Consortium, et al. (this issue). Consensus recommendations on Mental health issues in Phelan-McDermid syndrome.

van der Knaap, M.S., Boor, I., Estévez, R., 2012. Megalencephalic leukoencephalopathy with subcortical cysts: chronic white matter oedema due to a defect in brain ion and water homeostasis. Lancet Neurol. 11, 973–985. https://doi.org/10.1016/S1474-4422(12)70192-8.

Verma, R., Mukerji, M., Grover, D., B-Rao, C., Das, S.K., Kubendran, S., et al., 2005. MLC1 gene is associated with schizophrenia and bipolar disorder in southern India. Biol. Psychiatr. 58, 16–22. https://doi.org/10.1016/j.biopsych.2005.03.027.

Verpelli, C., Dvoretskova, E., Vicidomini, C., Rossi, F., Chiappalone, M., Schoen, M., et al., 2011. Importance of Shank3 protein in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses. J. Biol. Chem. 286, 34839–34850. https://doi.org/10.1074/jbc.M111.258384.

Vetrini, F., McKee, S., Rosenfeld, J.A., Suri, M., Lewis, A.M., Nugent, K.M., et al., 2019. De novo and inherited TCF20 pathogenic variants are associated with intellectual disability, dysmorphic features, hypotonia, and neurological impairments with similarities to Smith-Magenis syndrome. Genome Med. 11, 12. https://doi.org/10.1186/s13073-019-0623-0.

Wakazono, T., Miyake, M., Yamashiro, K., Yoshikawa, M., Yoshimura, N., 2016. Association between SCO2 mutation and extreme myopia in Japanese patients. Jpn. J. Ophthalmol. 60, 319–325. https://doi.org/10.1007/s10384-016-0442-4.

Walinga, M., Jesse, S., Alhambra, N., The European Phelan-McDermid Syndrome Consortium, Van Buggenhout, G. (this issue). Consensus recommendations on altered sensory function in Phelan-McDermid syndrome.

Warrier, V., Zhang, X., Reed, P., Havdahl, A., Moore, T.M., Cliquet, F., et al., 2022. Genetic correlates of phenotypic heterogeneity in autism. Nat. Genet. 54, 1293–1304. https://doi.org/10.1038/s41588-022-01072-5.

Wilson, H.L., Crolla, J.A., Walker, D., Artifoni, L., Dallapiccola, B., Takano, T., et al., 2008. Interstitial 22q13 deletions: genes other than SHANK3 have major effects on cognitive and language development. Eur. J. Hum. Genet. 16, 1301–1310. https://doi.org/10.1038/ejhg.2008.107.

Wong, M.-L., Arcos-Burgos, M., Liu, S., Vélez, J.I., Yu, C., Baune, B.T., et al., 2017. The PHF21B gene is associated with major depression and modulates the stress response. Mol. Psychiatr. 22, 1015–1025. https://doi.org/10.1038/mp.2016.174.

Xu, N., Lv, H., Yang, T., Du, X., Sun, Y., Xiao, B., et al., 2020. A 29 Mainland Chinese cohort of patients with Phelan-McDermid syndrome: genotype-phenotype correlations and the role of SHANK3 haploinsufficiency in the important phenotypes. Orphanet J. Rare Dis. 15, 1–12. https://doi.org/10.1186/s13023-020-01592-5.

Xue, J., Milburn, P.J., Hanna, B.T., Graham, M.E., Rostas, J.A.P., Robinson, P.J., 2004. Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals. Biochem. J. 381, 753–760. https://doi.org/10.1042/BJ20040455.

Yałcinkaya, C., Yüksel, A., Çomu, S., Kiliç, G., Çokar, Ö., Dervent, A., 2003. Epilepsy in vacuolating megalencephalic leukoencephalopathy with subcortical cysts. Seizure 12, 388–396. https://doi.org/10.1016/S1059-1311(02)00350-3.