# Microduplications of ARID1A and ARID1B cause a novel clinical and epigenetic distinct BAFopathy

Pleuntje J van der Sluijs, Sébastien Moutton, Alexander J M Dingemans, Denisa Weis, Michael A Levy, Kym M Boycott, Claudia Arberas, Margherita Baldassarri, Claire Beneteau, Alfredo Brusco, et al.

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Pleuntje J van der Sluijs, Sébastien Moutton, Alexander J M Dingemans, Denisa Weis, Michael A Levy, et al.. Microduplications of ARID1A and ARID1B cause a novel clinical and epigenetic distinct BAFopathy. Genetics in Medicine, 2025, 27 (1), pp.101283. 10.1016/j.gim.2024.101283.1. hal-05163561

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ARTICLE

# Microduplications of *ARID1A* and *ARID1B* cause a novel clinical and epigenetic distinct BAFopathy

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Pleuntje J. van der Sluijs¹ ID, Sébastien Moutton²,³, Alexander J.M. Dingemans⁴,
Denisa Weis⁵, Michael A. Levy⁶, Kym M. Boycott⁷,⁸, Claudia Arberas⁹,
Margherita Baldassarri¹⁰,¹¹, Claire Beneteau¹²,¹³,¹⁴, Alfredo Brusco¹⁵, Charles Coutton¹⁶,¹⁷,
Tabib Dabir¹⁸, Maria L. Dentici¹⁹, Koenraad Devriendt²⁰, Laurence Faivre³,²¹,
Mieke M. van Haelst²²,²³, Khadije Jizi²⁴,²⁵, Marlies J. Kempers⁴, Jennifer Kerkhof⁶,
Mira Kharbanda²⁶, Katherine Lachlan²⁶,²⁷, Nathalie Marle²⁸, Haley McConkey⁶,²⁹,
Maria A. Mencarelli¹⁰, David Mowat³⁰,³¹, Marcello Niceta³², Claire Nicolas³,²¹,
Antonio Novelli³³, Valeria Orlando³³, Olivier Pichon¹², Julia Rankin³⁴, Raissa Relator⁶,
Fabienne G. Ropers³⁵, Jill A. Rosenfeld³⁶,³⁷, Rani Sachdev³¹,³⁸, Sarah A. Sandaradura³⁹,⁴⁰,
Elena Shukarova-Angelovska⁴¹, Duco Steenbeek⁴², Marco Tartaglia³², Matthew A. Tedder⁴³,
Slavica Trajkova¹⁵,⁴⁴, Norbert Winer⁴⁵,⁴⁶, Jeremy Woods⁴⁷,⁴⁸, Bert B.A. de Vries⁴,
Bekim Sadikovic⁶,²⁹, Marielle Alders⁴⁹, Gijs W.E. Santen¹,*, ID

ARTICLE INFO

Article history:

Received 20 March 2024
Received in revised form
23 September 2024
Accepted 23 September 2024
Available online 28 September 2024

Keywords:

ARID1A
ARID1B
BAF complex
Duplication
Intellectual Disability

ABSTRACT

**Purpose:** *ARID1A/ARID1B* haploinsufficiency leads to Coffin-Siris syndrome, duplications of *ARID1A* lead to a distinct clinical syndrome, whilst *ARID1B* duplications have not yet been linked to a phenotype.

**Methods:** We collected patients with duplications encompassing *ARID1A* and *ARID1B* duplications.

**Results:** 16 *ARID1A* and 13 *ARID1B* duplication cases were included with duplication sizes ranging from 0.1 to 1.2 Mb (1-44 genes) for *ARID1A* and 0.9 to 10.3 Mb (2-101 genes) for *ARID1B*. Both groups shared features, with *ARID1A* patients having more severe intellectual disability, growth delay, and congenital anomalies. DNA methylation analysis showed that *ARID1A* patients had a specific methylation pattern in blood, which differed from controls and from patients with *ARID1A* or *ARID1B* loss-of-function variants. *ARID1B* patients appeared to have a distinct methylation pattern, similar to *ARID1A* duplication patients, but further research is needed to validate these results. Five cases with duplications including *ARID1A* or *ARID1B* initially annotated as duplications of uncertain significance were evaluated using PhenoScore and DNA methylation reanalysis, resulting in the reclassification of 2 *ARID1A* and 2 *ARID1B* duplications as pathogenic.

The Article Publishing Charge (APC) for this article was paid by Gijs W.E. Santen under an agreement between the LUMC and Elsevier.

*Correspondence and requests for materials should be addressed to Gijs Santen, Department of Clinical Genetics, Leiden University Medical Center, Albinusdreef 2, Postbus 9600, 2300 RC Leiden, The Netherlands. Email address: santen@lumc.nl

Affiliations are at the end of the document.
**Conclusion:** Our findings reveal that *ARID1B* duplications manifest a clinical phenotype, and *ARID1A* duplications have a distinct episignature that overlaps with that of *ARID1B* duplications, providing further evidence for a distinct and emerging BAFopathy caused by whole-gene duplication rather than haploinsufficiency.

© 2024 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

In the last decade, pathogenic germline variants in BAF complex genes have been frequently identified in patients with intellectual disability (ID),¹,² making it one of the most common causes of ID. *ARID1B* is the most frequently mutated gene in the BAF complex.³⁻⁷ *ARID1A* (HGNC:11110, NM_006015.6) and *ARID1B* (HGNC:18040, NM_020732.3) are mutually exclusive parts of the complex with a similar structure and function. Loss-of-function variants or deletions in either gene cause non-syndromic ID or Coffin-Siris syndrome (CSS)¹,³,⁵ (OMIM 13500 and 614607). These patients all have a BAFopathy episignature present in their blood.⁸

The *ARID1A* and *ARID1B* genes are both triple sensitive genes.⁹ Deletions or truncating variants in *ARID1A*, as well as *ARID1B* result in CSS, whereas *ARID1A* whole-gene duplications lead to a distinct clinical syndrome marked by intellectual disability, microcephaly and growth delay in most patients.¹⁰⁻¹² This suggests that *ARID1B* whole-gene duplications may lead to a similar phenotype; however, no patients with *ARID1B* duplications have been reported so far.

To investigate whether an *ARID1B* duplication phenotype exists, and how this relates to the *ARID1A* phenotype, we included patients with an *ARID1A* or *ARID1B* duplication, compared their (facial) phenotypes,¹³ and determined DNA methylation pattern⁸ in a subset of patients.

## Materials and Methods

### Patient collection

Patients were identified through various sources, including international contacts, physician referrals for second opinions, the DECIPHER¹⁴ database, and the Coffin-Siris International Facebook group. Furthermore, patients were also recruited through our outpatient CSS expertise center located at Leiden University Medical Center in the Netherlands.

### Data collection

Data were collected via an online questionnaire, which was completed by the referring physician.

## DNA methylation

DNA samples derived from peripheral blood were available from 15 patients, and DNA methylation data were generated using bisulfited-converted DNA and Illumina Infinium EPIC arrays. Analysis of the DNA methylation array data was performed at the Verspeeten Clinical Genome Centre in London, Canada, using the established previously described EpiSign episignature development pipeline.¹⁵⁻¹⁷ Data were normalized, beta values were generated, and cell composition was estimated using the R package sesame (version 1.18.4).¹⁸ Y chromosome probes, probes that contained SNPs at the CpG interrogation or single-nucleotide extension sites, and probes which are known to cross-react with other genomic locations were removed.¹⁹ For each analysis a set of control samples was chosen from the EpiSign Knowledge Database, consisting of disorder-specific samples¹⁷,²⁰ and unaffected controls. Controls included samples from the case batches and from other batches and were matched for sex and age using the R package MatchIt (version 4.5.4). Beta values were converted to M values for linear modeling. Using the R package limma (version 3.56.2), a linear regression model with estimated blood cell composition as a covariate was fitted, and the empirical Bayes method was used to calculate moderated t-statistics and corresponding *P* values. *P* values were adjusted using the Benjamini-Hochberg method to control for false discovery rates. Microarray probes with a mean difference between cases and controls of less than 5% were removed. Probe selection was performed using a combination of effect size and *P* values (absolute methylation difference multiplied by -log(*P* value)), receiver operating characteristic curve analysis, and removal of highly correlated probes, with parameter values selected that maximized the differences between cases and controls as assessed using hierarchical clustering and multidimensional scaling. A support vector machine classifier was created using the R package e1071 (version 1.7-13). To increase specificity for the ARID1 classifiers, the models were trained not just against the matched controls but also against additional controls and disorder-specific samples from the EpiSign Knowledge Database. Support vector machine decision values were converted to probability scores according to the Platt's scaling method. Leave-one-out cross-validation was performed for each case sample and evaluated using hierarchical clustering, multidimensional scaling, and methylation variant pathogenicity scores.
## PhenoScore

Quantitative phenotypic similarity analysis was performed with PhenoScore. PhenoScore is a machine learning framework that combines facial recognition technology with Human Phenotype Ontology (HPO)-based phenotype analyses to quantify phenotypic similarity.²¹ These analyses were performed as previously described using clinical data of 14 patients with *ARID1A* (9 with facial photographs) and 10 patients *ARID1B* whole-gene duplications (4 with facial photographs). The following comparisons were performed: (1) whether patients were distinguishable from age-, sex-, and ethnicity-matched neurodevelopmental (NDD) controls; (2) whether *ARID1A* duplication patients were distinguishable from *ARID1B* duplication patients; and (3) whether ARID1 whole-gene duplication patients were recognizable from *ARID1A* and *ARID1B* patients with loss-of-function variants. NDD-matched controls were selected from the Radboud University Medical Center inhouse control database, which contains phenotypic data (both facial and other clinical features in HPO) of 1600 individuals with NDD. *P* values <.05 were considered significant. For each duplication of *ARID1A* or *ARID1B* initially interpreted as a duplication of uncertain significance several PhenoScore analyses were performed. It was determined whether the patient data (ie, HPO-terms) and, when available, facial photos clustered with HPO-terms and photos of ARID1 duplication patients, controls, or patients with loss-of-function variants in *ARID1A* and *ARID1B*.

## Interpretation of duplications of uncertain significance

After DNA methylation and PhenoScore analyses of variants with whole-gene duplications, the variants initially classified as a duplication of uncertain significance either because of the size of the duplication or based on the phenotypic assessment of the clinician, were interpreted using the American College of Medical Genetics and Genomics criteria for copy-number variants.²² American College of Medical Genetics and Genomics-criteria 4L “Statistically significant increase among observations in cases (with a consistent, specific, well-defined phenotype) compared with controls” was allocated when a specific DNA methylation pattern was identified in a patient with a duplication of uncertain significance.

## Genotype-phenotype description

Only data of clearly pathogenic variants were included in the phenotype overview and PhenoScore analysis. All analyses were executed using SPSS version 25. R version 4.1.0 was used to create graphs.

## Results

### Genotype

We identified 14 patients with a whole-gene duplication of *ARID1A* and 2 patients with a duplication of uncertain significance in *ARID1A* either due to the size of the duplication or based on the estimation of the clinician based on the presenting phenotype. In addition, 11 patients with a whole-gene duplication of *ARID1B* and 5 patients (including 2 parents) with a duplication of uncertain significance in *ARID1B* were identified (Supplemental Table 1). The size of the duplications ranged from 0.1 to 1.2 Mb with 1 to 44 genes for *ARID1A* and 0.9 to 10.3 Mb with 2 to 101 genes for *ARID1B* (Figure 1). Inheritance was tested in 24 cases. All whole-gene duplications, except 3 (all *ARID1B*), occurred de novo. In case 5.1B the duplication was the result of a maternal balanced translocation, whereas in the other cases, V1i_1.1B and V2_1.1B, the duplication was inherited from a potentially affected parent. We also included 3 cases with partial duplications of uncertain significance.

To investigate whether there were any other potential disease-causing genes included in the duplicated regions, we searched the maximal duplicated region in the DECIPHER database¹⁴ and ordered the genes in this region in descending order of pTriplo. For the chromosome 1p36, region *ARID1A* was the top result, and only 1 other gene (ie, *WDTC1* [HGNC:29175]) had a score >0.95. This gene has not been linked to a human phenotype and is present in the duplication of patient 4.1A only and therefore is unlikely to influence our results.

For chromosome 6q25 *ARID1B* was the 2nd result after *SYNE1* (HGNC:17089), which is duplicated in 3 out of 13 patients (ie, 1.1B, 2.1B, and 3.1B). Homozygous *SYNE1* variants have been linked to arthrogryposis phenotype (OMIM 618484) and spinocerebellar ataxia (OMIM 610743), while heterozygous variants can cause Emery-Dreifuss muscular dystrophy (OMIM 612998). Duplications, however, have not been previously associated with human disease. The only other gene with a pTriplo score >0.95 was *SCAF8* (HGNC:20959), which is duplicated in 4/13 *ARID1B* patients (ie, 1.1B, 2.1B, 3.1B, and 4.1B) and has not been linked to a human phenotype. *SCAF8* and *SYNE1* are duplicated in only those *ARID1B* patients with the largest duplications. Our analysis of *ARID1B* duplication size showed no differences in severity or frequency of congenital anomalies, although patient numbers remain low. We therefore tentatively conclude that, based on current knowledge, *ARID1A* and *ARID1B* are the primary genes of interest in the duplicated regions of our patients.

In 3 patients other small duplications or a deletions were identified, of which 2 were deemed nonpathogenic. Patient 7.1A also carried 2 inherited deletions at 8p23.1 and 9p24.1. Both deletions were identified in nonaffected parents and/or
<figure><img src="image_4.png" /><figcaption>**Figure 1** Genomic rearrangements in patients with *ARID1A* and *ARID1B* duplications. Alignment in the University of California, Santa Cruz (UCSC) genome browser of the genomic rearrangements in our patients with gene duplications of (A) *ARID1A* and (B) *ARID1B*. The red square indicates the length of the transcript of the gene.</figcaption></figure>

siblings. Patient 2.1B also carried a 5q35.3 duplication. Based on previous findings this duplication is unlikely to be pathogenic.²³ Patient 7.1B carries a 3p26.3 duplication (NC_000003.11:g.(?_225645)_(1510822_?)dup) including *CHL1* (HGNC:1939) and *CNTN6* (HGNC:2176). Sporadic cases of a 3p26.3 duplications have been reported²⁴⁻²⁶; therefore, this variant may have contributed to this patient's phenotype.

## DNA methylation

We performed methylation analysis in DNA samples (all isolated from peripheral blood) of 15 cases. In 5 patients with a whole-gene duplication of *ARID1A*, the DNA methylation pattern detected was found to be distinct from controls (Figure 2A and B, Supplemental Figure 1A, Supplemental Table 2). This distinct pattern was opposite to the pattern observed in patients with more common loss-of-function variants of *ARID1A* or *ARID1B* (Figure 2,

Supplemental Figure 1D, G, and F). Two cases with variant of uncertain significance (V1.1A and V2i.1A) clustered with cases with *ARID1A* whole-gene duplications, supporting their pathogenicity. DNA methylation analyses in patients with whole-gene duplication of *ARID1B* showed that these cases clustered together and separate from controls (Figure 2C and D, Supplemental Table 2). Unfortunately, cross-validation failed for this potential episignature (Supplemental Figure 1B). The *ARID1B* duplication of uncertain significance of exon 6-20 (V1i_1.1B) clustered with cases and not with controls. The intragenic quadruplication of exon 6-10 of *ARID1B* (V3i.1B) clustered, as hypothesized beforehand, with BAFopathy samples, supporting its pathogenicity as a loss-of-function variant.

When clustering patient samples using the *ARID1A* duplication probes, *ARID1A* duplication cases cluster separately from controls and form a separate cluster within the BAFopathy samples. *ARID1B* duplication samples cluster scattered through the controls (except for V3i.1B with a BAFopathy episignature) (Supplemental Figure 1C and D).
<figure><img src="image_5.png" /><figcaption>**Figure 2** Hierarchical clustering with heatmap of methylation data. Clustering analysis was performed using Ward's method on Euclidean distance. Rows represent the CpG probes, and columns represent the participants. The color scale from dark blue to dark red represents the range of the methylation levels (beta values) between 0 and 1. The legend represents the pathogenicity interpretation before DNA methylation analysis, ie, pathogenic (red), likely pathogenic (orange), variant of uncertain significance (VUS) (purple), or control (green). Hierarchical clustering of 200 probes differentially methylated between *ARID1A* whole-gene duplication patients and controls generates 2 clusters: one composed of all *ARID1A* patients with pathogenic and the other composed of controls (A). A similar pattern is</figcaption></figure>
Interestingly, when using the *ARID1B* duplication probes all *ARID1A* and *ARID1B* duplication cases (except for V3i.1B) cluster together, separate from controls and BAFopathy samples (Supplemental Figure 1E and F). Using the BAFopathy probes, all duplication samples (except V3i.1B) clustered within controls (Supplemental Figure 1G and H).

## PhenoScore analysis of variants of uncertain significance

Our study included several duplications of uncertain significance. We used PhenoScore to analyze whether they clustered with ARID1 duplication cases, CSS patients, or NDD control cases based on their HPO terms and/or facial photographs, if available. Case V1i_1.1B, with a partial *ARID1B* duplication of exon 6-20, clustered with CSS cases. Case V2i.1A, with a partial *ARID1A* duplication of exon from halfway exon 1-20, clustered with CSS cases, indicating that the phenotype of this individual is more similar to the phenotypes of those patient groups than of NDD controls. Interestingly, case V3i.1B clustered with all patient groups and not with controls, indicating that the phenotype is more similar to both CSS and ARID1 dup cases compared with NDD controls. Case V1.1.A with an *ARID1A* whole-gene duplication has a high phenotypic similarity score (0.71) only to individuals with *ARID1A* duplications (Supplemental Table 4). Patient V1_1.1B and her father (V1_2.1B) both with an *ARID1B* whole-gene duplication did not cluster with CSS cases, whereas V1_2.1B was seen as more phenotypically similar to *ARID1B* duplication cases. Adding facial photographs did not substantially change these results (Supplemental Table 5).

## Interpretation of duplications of uncertain significance

Both *ARID1A* duplications of uncertain significance (ie, V1.1A, V2i.1A) were reclassified as pathogenic *ARID1A* duplications. Two of the 3 unique *ARID1B* duplications of uncertain significance were classified as pathogenic. Patients V2_1.1B and V2_2.1B were regarded as carrying a pathogenic *ARID1B* whole-gene duplication, whereas case V3i.1B (with a de novo intragenic triplication of *ARID1B*) was considered to have CSS. The inherited duplication of exon 6-20 of *ARID1B* in patients V1i_1.1B and V1i_2.1B was considered to remain a duplication of uncertain significance (Supplemental Table 1).

## Phenotype

Clinical data were obtained for 16 cases with a pathogenic *ARID1A* duplication and 13 cases with a pathogenic *ARID1B* duplication (Table 1, Supplemental Figure 2),

detailed information was available of 14 *ARID1A* and 11 *ARID1B* cases. In case 3.1B with intrauterine growth restriction (IUGR), severe polyhydramnios, and an *ARID1B* duplication of 6.3 Mb, the pregnancy was terminated. For all other cases, the age at last examination ranged from 1 to 46 years for patients with *ARID1A* duplications and from 1.5 to 31 years for patients with *ARID1B* duplications.

## ARID1A and ARID1B

*ARID1A* duplication patients exhibited more severe developmental delays and intellectual disability (ID) compared with *ARID1B* duplication patients, as evidenced by their developmental milestones and IQ scores (Figure 3, Table 1). Head circumference tended to be smaller in *ARID1A* duplication patients compared with *ARID1B* duplication patients (Figure 3D, Table 2). Feeding difficulties were more common among *ARID1A* patients, with a prevalence of 67% (8/12) compared with 20% (1/5) in *ARID1B* patients.

## Duplication size

After correcting for the affected gene, the analyses comparing patients with smaller duplications (*ARID1A*: *n* = 8, *ARID1B*: *n* = 6) to those with larger duplications (more than 5 genes) revealed that there is no difference between patients with duplications including more than 5 genes compared with patients with 5 or less genes in the duplicated region in motor and speech development, congenital anomalies or seizures (Supplemental Figure 3).

## Shared features

Patients with an *ARID1A* or *ARID1B* duplication share several specific features, including intellectual disability (14/14 *ARID1A*, 10/11 *ARID1B*), growth delay (10/14 *ARID1A*, 4/10 *ARID1B*), cryptorchidism (*n* = 5 *ARID1A*, *n* = 3 *ARID1B*), and pes planus (6/8 *ARID1A*, 1/5 *ARID1B*) (Table 2).

## PhenoScore analysis

The PhenoScore analysis indicated that *ARID1A* duplication patients more often tend to have retractile testis, overweight, low birth weight, short stature, and microcephaly compared with controls (Supplemental Figure 4A and B). *ARID1B* duplication patients tend to have more motor delay, retractile testes, mild-moderate ID, and less joint hypermobility compared with controls (Supplemental Figure 4C and D). Combining the data from *ARID1A* and *ARID1B* duplication patients and comparing them with controls produces results comparable to those obtained from separate analyses (Supplemental Figure 4E and F). Comparing *ARID1A* with *ARID1B* duplication patients, *ARID1A* duplication patients more frequently tend to have constipation, IUGR, reflux, and pes planus, whereas *ARID1B* patients more frequently tend to have mild-moderate ID, myopia, and retractile testis (Supplemental Figure 4G). The PhenoScore analysis also

observed using the 219 probes that were identified for the *ARID1B* whole-gene duplication samples; in this case 1 VUS (patient V3i.1B) clusters with controls (B). MDS plots showing a similar the differentiation as in the heatmap between cases and controls for *ARID1A* (C) and *ARID1B* (D) cases.
<table><caption>**Table 1** Clinical characteristics of patients with *ARID1A* or *ARID1B* whole-gene duplication</caption><thead><tr><th rowspan="2">Clinical and Developmental Findings</th><th colspan="3">ARID1A, n = 16<sup>a</sup></th><th colspan="3">ARID1B, n = 13<sup>b</sup></th><th rowspan="2">P Value</th><th rowspan="2">Test</th></tr><tr><th>Affected</th><th>Total</th><th>%</th><th>Affected</th><th>Total</th><th>%</th></tr></thead><tbody><tr><td>Sex, female</td><td>7</td><td>16</td><td>44%</td><td>5</td><td>13</td><td>38%</td><td>1.00</td><td>χ<sup>2</sup></td></tr><tr><td>Neurodevelopmental delay</td><td>13</td><td>13</td><td>100%</td><td>7</td><td>7</td><td>100%</td><td></td><td></td></tr><tr><td>Intellectual disability</td><td>14</td><td>14</td><td>100%</td><td>10</td><td>11</td><td>91%</td><td>.03</td><td>Fisher's</td></tr><tr><td>Mild</td><td>1</td><td>14</td><td>7%</td><td>6</td><td>11</td><td></td><td></td><td></td></tr><tr><td>Moderate</td><td>6</td><td>14</td><td>43%</td><td>3</td><td>11</td><td></td><td></td><td></td></tr><tr><td>Severe</td><td>6</td><td>14</td><td>43%</td><td>1</td><td>11</td><td></td><td></td><td></td></tr><tr><td>Profound</td><td>1</td><td>14</td><td>7%</td><td>0</td><td>11</td><td></td><td></td><td></td></tr><tr><td>Language delay</td><td>15</td><td>15</td><td>100%</td><td>9</td><td>9</td><td>100%</td><td></td><td></td></tr><tr><td>No words</td><td>4</td><td>12</td><td>33%</td><td>0</td><td>8</td><td>0%</td><td>.12</td><td>Fisher's</td></tr><tr><td>Brain anomaly<sup>c</sup></td><td>4</td><td>11</td><td>36%</td><td>1</td><td>4</td><td>25%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Behavior anomaly</td><td>9</td><td>13</td><td>69%</td><td>6</td><td>7</td><td>86%</td><td>.61</td><td>Fisher's</td></tr><tr><td>Short attention span</td><td>4</td><td>13</td><td>31%</td><td>2</td><td>7</td><td>29%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Autistic traits</td><td>2</td><td>13</td><td>15%</td><td>3</td><td>7</td><td>43%</td><td>.29</td><td>Fisher's</td></tr><tr><td>Stereotyped behavior</td><td>3</td><td>13</td><td>23%</td><td>1</td><td>7</td><td>14%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Epilepsy/seizures</td><td>1</td><td>11</td><td>9%</td><td>2</td><td>7</td><td>29%</td><td>.72</td><td>Fisher's</td></tr><tr><td>Vision impaired</td><td>6</td><td>11</td><td>55%</td><td>5</td><td>7</td><td>71%</td><td>.64</td><td>Fisher's</td></tr><tr><td>Myopia</td><td>1</td><td>4</td><td>25%</td><td>3</td><td>4</td><td>75%</td><td>.49</td><td>Fisher's</td></tr><tr><td>Hypermetropia</td><td>2</td><td>4</td><td>50%</td><td>2</td><td>3</td><td>67%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Hearing impaired</td><td>3</td><td>9</td><td>33%</td><td>1</td><td>5</td><td>20%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Pregnancy anomaly<sup>c</sup></td><td>7</td><td>13</td><td>54%</td><td>4</td><td>10</td><td>40%</td><td>.68</td><td>Fisher's</td></tr><tr><td>Growth delay</td><td>10</td><td>14</td><td>71%</td><td>4</td><td>10</td><td>40%</td><td>.21</td><td>Fisher's</td></tr><tr><td>Birthweight (SDS)</td><td>mean -1.4, SDS 1.1</td><td>n = 12</td><td></td><td>mean -0.07, SDS 1.1</td><td>n = 7</td><td></td><td>.02</td><td>t test</td></tr><tr><td>BW &lt;-2SDS</td><td>4</td><td>12</td><td>33%</td><td>0</td><td>7</td><td>0%</td><td>.25</td><td>Fisher's</td></tr><tr><td>OFC at birth (SDS)</td><td>mean -1.6, SDS 0.4</td><td>n = 3</td><td></td><td>mean 0.3, SDS 1.6</td><td>n = 3</td><td></td><td>.11</td><td>t test</td></tr><tr><td>Height (SDS)</td><td>mean -2.2, SDS 1.3</td><td>n = 12</td><td></td><td>mean -0.1, SDS 2.1</td><td>n = 6</td><td></td><td>&lt;.01</td><td>t test</td></tr><tr><td>Height (&lt;-2 SDS)</td><td>8</td><td>12</td><td>67%</td><td>1</td><td>6</td><td>17%</td><td>.13</td><td>Fisher's</td></tr><tr><td>Weight (SDS - weight for length)</td><td>mean 0.8, SDS 2.6</td><td>n = 11</td><td></td><td>mean 1.4, SDS 1.6</td><td>n = 5</td><td></td><td>.32</td><td>t test</td></tr><tr><td>Weight (&gt;2 SDS - weight for length)</td><td>5</td><td>11</td><td>45%</td><td>2</td><td>5</td><td>40%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>OFC (SDS)</td><td>mean -2.7, SDS 1.2</td><td>n = 12</td><td></td><td>mean -1.0, SDS 2.4</td><td>n = 6</td><td></td><td>.06</td><td>t test</td></tr><tr><td>OFC (&lt;-2 SDS)</td><td>11</td><td>15</td><td>73%</td><td>2</td><td>6</td><td>33%</td><td>.15</td><td>Fisher's</td></tr><tr><td>Ectodermal anomalies</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Nail anomalies</td><td>3</td><td>12</td><td>25%</td><td>1</td><td>5</td><td>20%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Abnormal dentition</td><td>3</td><td>9</td><td>33%</td><td>3</td><td>6</td><td>50%</td><td>.62</td><td>Fisher's</td></tr><tr><td>Facial characteristics</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Prominent/low columella</td><td>3</td><td>8</td><td>38%</td><td>1</td><td>4</td><td>25%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Short philtrum</td><td>5</td><td>10</td><td>50%</td><td>0</td><td>6</td><td>0%</td><td>.09</td><td>Fisher's</td></tr><tr><td>Thin upper lip</td><td>9</td><td>11</td><td>82%</td><td>3</td><td>5</td><td>60%</td><td>.48</td><td>Fisher's</td></tr><tr><td>Cleft palate</td><td>1</td><td>8</td><td>13%</td><td>2</td><td>9</td><td>22%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Small mouth</td><td>4</td><td>6</td><td>67%</td><td>0</td><td>4</td><td>0%</td><td>.08</td><td>Fisher's</td></tr><tr><td>Ear anomalies</td><td>5</td><td>12</td><td>42%</td><td>2</td><td>6</td><td>33%</td><td>1.00</td><td>Fisher's</td></tr></tbody></table>

(continued)
<table><caption>**Table 1** Continued</caption><thead><tr><th rowspan="2">Clinical and Developmental Findings</th><th colspan="3">ARID1A, n = 16<sup>a</sup></th><th colspan="3">ARID1B, n = 13<sup>b</sup></th><th rowspan="2">P Value</th><th rowspan="2">Test</th></tr><tr><th>Affected</th><th>Total</th><th>%</th><th>Affected</th><th>Total</th><th>%</th></tr></thead><tbody><tr><td><strong>Skeletal anomalies</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Hand and foot anomalies</td><td>12</td><td>12</td><td>100%</td><td>3</td><td>5</td><td>60%</td><td>.07</td><td>Fisher's</td></tr><tr><td>Hand anomaly</td><td>9</td><td>10</td><td>90%</td><td>2</td><td>5</td><td>40%</td><td>.08</td><td>Fisher's</td></tr><tr><td>Foot anomaly</td><td>7</td><td>11</td><td>64%</td><td>2</td><td>5</td><td>40%</td><td>.60</td><td>Fisher's</td></tr><tr><td>Pes planus</td><td>6</td><td>8</td><td>75%</td><td>1</td><td>5</td><td>20%</td><td>.10</td><td>Fisher's</td></tr><tr><td>Spinal malformations</td><td>2</td><td>13</td><td>15%</td><td>1</td><td>9</td><td>11%</td><td>1.00</td><td>Fisher's</td></tr><tr><td>Joint laxity</td><td>3</td><td>7</td><td>43%</td><td>3</td><td>5</td><td>60%</td><td>1.00</td><td>Fisher's</td></tr><tr><td><strong>Cardiovascular anomalies<sup>c</sup></strong></td><td>3</td><td>12</td><td>25%</td><td>1</td><td>6</td><td>17%</td><td>1.00</td><td>Fisher's</td></tr><tr><td><strong>Gastrointestinal anomalies</strong></td><td>10</td><td>13</td><td>77%</td><td>3</td><td>7</td><td>43%</td><td>.17</td><td>Fisher's</td></tr><tr><td>Feeding problems</td><td>8</td><td>12</td><td>67%</td><td>1</td><td>5</td><td>20%</td><td>.13</td><td>Fisher's</td></tr><tr><td>GERD</td><td>5</td><td>13</td><td>38%</td><td>0</td><td>7</td><td>0%</td><td>.11</td><td>Fisher's</td></tr><tr><td>Constipation</td><td>6</td><td>13</td><td>46%</td><td>1</td><td>7</td><td>14%</td><td>.33</td><td>Fisher's</td></tr><tr><td><strong>Recurrent airway infections</strong></td><td>5</td><td>12</td><td>42%</td><td>0</td><td>5</td><td>0%</td><td>.25</td><td>Fisher's</td></tr><tr><td><strong>Cryptorchidism</strong></td><td>5</td><td>6</td><td>83%</td><td>3</td><td>5</td><td>60%</td><td>.55</td><td>Fisher's</td></tr></tbody></table>

IQ, intellectual quotient; GERD, gastroesophageal reflux disease; OFC, occipital head circumference; SDS, standard deviation score.

<sup>a</sup>1 patient DECIPHER data only.

<sup>b</sup>1 case was a pregnancy termination.

<sup>c</sup>Reported pregnancy anomalies: polyhydramnios (1× ARID1A, 1× ARID1B), intrauterine growth retardation (3× ARID1A, 2× ARID1B), cardiac defect (1× ARID1A, 1× ARID1B). Reported brain anomalies: T2 hyperintensity and T1 hypointensity in the right thalamus, prominent cerebral cortical sulci, ventricles and extra axial cerebral spinal fluid spaces, cavum septum pellucidum (1× ARID1A), ventriculomegaly (3× ARID1A), delayed myelination (1× ARID1A), hypoplasia of the corpus callosum (1× ARID1A), dysmorphic brain particularly affecting the left hippocampus (1× ARID1B). Reported cardiovascular anomalies: pulmonary stenosis (1× ARID1A). An ARID1A patient had a atrium septum defect, muscular small ventricular septum defect (VSD), and mild-moderate valvular pulmonary stenosis (PS), concluded as a combination of small cardiac abnormalities without hemodynamic consequences. He also had an additional anatomical variation in division of arch vessels (left arch joint origin of brachiocephalic trunk and left carotid artery). The VSD closed spontaneously. PS was not important. Other reported anomalies in patients were VSD (2× ARID1A), and cardiac rhythm anomaly (1× ARID1B).
<figure><img src="image_6.png" /><figcaption>**Figure 3** Intellectual disability degree and developmental milestones of the *ARID1A* and *ARID1B* duplication patients. Histogram of the (A) distribution of intellectual disability severity, (B) height (SDS), (C) weight (SDS weight-for-height) and, (D) head circumference (SDS). Cumulative distribution of developmental milestones (E) sitting (n = 13), (F) walking (n = 15), and (G) first words (n = 11).</figcaption></figure>

revealed that, based on HPO terms, *ARID1A* and *ARID1B* duplication patients clustered just as far from each other as from NDD controls (PhenoScore 0.50) or CSS patients (PhenoScore of 0.52) (Supplemental Table 3).

## Discussion

In total, we report data from 16 patients with a pathogenic *ARID1A* and 13 patients with a pathogenic *ARID1B* gene duplication.

## Genotype-phenotype

Our study reveals a distinct clinical phenotype resulting from *ARID1B* whole-gene duplications, which shares features with the phenotype of *ARID1A* whole-gene duplications, including mild to severe intellectual disability, growth delay, and hand or feet anomalies, occasionally accompanied by a unique facial appearance.

Genes associated with the BAF complex have been linked to CSS, with each gene having its own genotype-phenotype association within the CSS-spectrum.²⁷,²⁸
<figure><img src="image_7.png" /><figcaption>**Figure 3** Continued.</figcaption></figure>

Patients with loss of function variants in *ARID1A* and *ARID1B* have clinical features most similar to each other within this spectrum, with *ARID1A* patients generally being more severely affected compared with those with pathogenic variants in *ARID1B*.⁶,⁷,²⁹⁻³¹ For duplications, we now also show that *ARID1A* duplications lead to a more severe phenotype than *ARID1B* duplications, considering that growth and ID-associated features differed significantly (Figure 3, Table 1). However, the PhenoScore analysis (Supplemental Figure 4, Supplemental Table 3), DNA methylation (Figure 2, Supplemental Figure 1), and facial photographs (Supplemental Figure 2) suggest that the

duplications of *ARID1A* and *ARID1B* are not merely differing in severity, but should be considered different clinical entities.

**CSS patients compared with whole-gene duplication patients**

Loss-of-function variants, including deletions in *ARID1A* and *ARID1B*, result in CSS. CSS is characterized by ID, feeding difficulties, congenital anomalies, hypoplasia of the fifth digits and/or nails, hypertrichosis, and distinctive facial features. Nonspecific features, such as ID, feeding difficulties, and congenital anomalies, were also present in our
<figure><img src="image_8.png" /><figcaption>Figure 3 Continued.</figcaption></figure>

patients with whole-gene duplications. However, CSS-specific features, such as shirt fifth digit, hypoplasia of the nail of a fifth digit, hypertrichosis, and the distinct facial features of CSS, were not observed. This is reflected by the PhenoScore analysis showing that *ARID1A* and *ARID1B* patients with loss-of-function variants including deletions cluster separately from *ARID1A* and *ARID1B* whole-gene duplication patients (Supplemental Table 3).

## DNA methylation

Over the last several years, researchers have discovered a growing number of unique DNA methylation patterns associated with specific disorders, referred to as episignatures.²⁰ These episignatures have become valuable biomarkers in the diagnostic testing for these disorders,¹⁷,³² many of which are linked to chromatin remodeling genes, including genes that encode components of the BAF complex. In our study, we utilized DNA methylation analysis to explore the impact of *ARID1A* and *ARID1B* whole-gene duplications on these episignatures. Interestingly, we found a specific *ARID1A* duplication DNA episignature and the determined the presence of an *ARID1B* duplication episignature. It has been discussed that an intermediate clustering of a sample or episignature may reflect an intermediate phenotype.³³ It could therefore be that the inconclusive scoring of the DNA methylation data of *ARID1B* duplication patients is due to their milder phenotype. Using the *ARID1B* duplication probes the *ARID1A* and *ARID1B*

duplication samples do appear to cluster together and separate from controls. The duplication episignatures differ from both controls and the episignatures of patients with other BAFopathies such as CSS/NCBRS, SMARCA2 BIS, SMARCA4, ARID2, SOX11, and others. All of this provides further evidence for the pathogenicity of *ARID1B* whole-gene duplications and suggests that *ARID1A* and *ARID1B* duplications cause a distinct BAFopathy.

The identification of a distinct DNA methylation pattern, or episignature, in patients with *ARID1A* whole-gene duplications provides clinicians with an additional tool to aid the interpretation of these duplications. For example, in case V2i.1A, the *ARID1A* exon 1 to exon 20 duplication, the *ARID1A* duplication DNA methylation pattern confirmed the pathogenicity of this variant. Whereas PhenoScore analysis showed an inconclusive clustering result. PhenoScore works for syndromic cases, a possible explanation could be that the features identified in our *ARID1A* duplication patients are too general (ID, growth delay, and microcephaly) or that there are too few patients in the discovery cohort. On the other hand, cases V1.1A and V2_2.1B, who had a *ARID1A* or *ARID1B* whole-gene duplication, who were classified as duplications of uncertain significance before DNA methylation results, tended to cluster with *ARID1A* or *ARID1B* duplication cases according to the PhenoScore analysis. After the final DNA methylation results these samples were reclassified as (likely) pathogenic. In our first attempts to identify an ARID1 duplication episignature using DNA methylation
<table><caption>**Table 2** Recommendations surveillance guidelines *ARID1A/ARID1B* duplication patients</caption><thead><tr><th rowspan="2">Evaluation (Inquire/Perform Physical Examination)</th><th rowspan="2">After Diagnosis</th><th colspan="3">Age Category</th><th rowspan="2">(Para)Medic<sup>a</sup></th></tr><tr><th>In Children (0-18 Years)</th><th>In Adults (18+)</th><th>Frequency</th></tr></thead><tbody><tr><td>Congenital abnormalities (heart, kidneys, and cryptorchidism)</td><td>Yes</td><td>Upon indication</td><td></td><td>Upon indication</td><td>treating physician</td></tr><tr><td>Feeding difficulties</td><td>Yes</td><td>Yes</td><td></td><td>At every visit</td><td>pediatrician</td></tr><tr><td>Constipation</td><td>Yes</td><td>Yes</td><td>Yes</td><td>At every visit</td><td>treating physician</td></tr><tr><td>Growth and weight</td><td>Yes</td><td>Yes</td><td>Yes</td><td>At every visit, to avoid excessive weight gain</td><td>pediatrician</td></tr><tr><td>Seizures</td><td>Yes</td><td>Yes</td><td>Yes</td><td>At every visit</td><td>treating physician</td></tr><tr><td>Endocrine/hormonal</td><td>On indication</td><td>Upon indication</td><td>Upon indication</td><td>Upon indication<sup>b</sup></td><td>treating physician</td></tr><tr><td>Vision</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Every 2 years during childhood, frequency can be adjusted if the patient is able to report on eyesight. In adults evaluation at age 30 and from age 40 every 5 years</td><td>ophthalmologist</td></tr><tr><td>Hearing</td><td>Yes</td><td>Yes</td><td>Upon indication</td><td>At every visit ask for signs of hearing loss and refer to audiologist if suspected</td><td>treating physician</td></tr><tr><td>Scoliosis</td><td>Yes</td><td>Yes</td><td></td><td>Periodically, until length growth is complete</td><td>orthopedic surgeon/ physiatrist/ pediatrician</td></tr><tr><td>Pedes (plano)valgi</td><td>Yes</td><td>Yes</td><td>On indication</td><td>Periodically</td><td>orthopedic surgeon/ physiatrist/ pediatrician</td></tr><tr><td>Dentition/dental health</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Twice a year</td><td>dentist</td></tr><tr><td>Motor development</td><td>If aged &lt;16</td><td>Yes</td><td></td><td>At every visit</td><td>pediatrician/ physiatrist/ physical therapist</td></tr><tr><td>Cognitive development</td><td>If aged &lt;16</td><td>Yes</td><td></td><td>At every visit</td><td>pediatrician/ psychologist</td></tr><tr><td>Communication/language development</td><td>Yes</td><td>Yes</td><td></td><td>Screening at age 2 and 3 years, then on indication</td><td>pediatrician/ physiatrist/ speech therapist</td></tr><tr><td>Behavioral and social development/ impairments</td><td>Yes</td><td>Yes</td><td>Yes</td><td>At every visit</td><td>treating physician</td></tr><tr><td>Sexual development</td><td>When applicable</td><td>Yes</td><td>Yes</td><td>When applicable</td><td>treating physician</td></tr><tr><td>Transition to adult care</td><td>When applicable</td><td>When applicable</td><td></td><td>At least from the age of 16 years</td><td>physiatrist/ pediatrician</td></tr></tbody></table>

<footnote><sup>a</sup>Treating physician (eg, general practitioner, pediatrician).</footnote>

<footnote><sup>b</sup>Especially, glucose, and thyroid lab.</footnote>
data from 8 *ARID1A* and 5 *ARID1B* patients, several *ARID1B* cases clustered with *ARID1A* cases, showing the overlap between the 2 episignatures (Figure 2, Supplemental Figure 1). Later findings unveiled distinctions in the DNA methylation patterns of patients with *ARID1A* and *ARID1B* duplications. This underscores the potential uncertainty associated with episignatures. Therefore, it is crucial to acknowledge that, although the presence of an episignature strongly indicates pathogenicity, its absence, although suggestive, does not definitively exclude it.

Similarly, PhenoScore can be useful in interpreting genetic variants with uncertain significance by providing further confirmation of their pathogenicity or non-pathogenicity. If a variant has a low suspicion of pathogenicity and lacks a DNA methylation episignature or clustering with cases, PhenoScore can offer additional evidence that the variant is nonpathogenic. Alternatively, PhenoScore can provide evidence for its pathogenicity, if a patient with such a variant clusters with cases (eg, V1.1A). However, it should be noted that PhenoScore, just like the *ARID1B* duplication episignature, is currently only used for research purposes, and its reliability needs to be further evaluated in clinical studies.

## Limitations

Our study has several limitations. First, our data were contributed by multiple clinicians and may not be complete in all cases. In addition, our cohort is predominantly composed of young patients, potentially leading to an underestimation of features that develop with age. Although we performed survival analyses to correct for this, further research including older adults is needed to gain a more complete understanding of the spectrum of features associated with advancing age.

## Conclusion

Our data reveal a distinct type of BAFopathy entity, characterized by a whole-gene duplication of *ARID1A* or *ARID1B*, rather than haploinsufficiency. The overlap in clinical phenotypes and episignatures between *ARID1B* and *ARID1A* whole-gene duplications supports the distinct nature of this BAFopathy.

Based on the clinical data of our cohort, we are suggesting the screening recommendations as described in Table 2.

## Data Availability

Deidentified patient data will be made available on request to the corresponding author.

## Acknowledgments

The authors would like to thank Johan den Dunnen for his assistance in adding patient data to the Leiden Open Variant Database.

This study makes use of data generated by the DECI-PHER community. A full list of centers who contributed to the generation of the data is available from [https://deciphergenomics.org/about/stats](https://deciphergenomics.org/about/stats) and via email from [contact@deciphergenomics.org](mailto:contact@deciphergenomics.org). Funding for the DECI-PHER project was provided by Wellcome.

Results from this article have been previously presented at the 55th European Society of Human Genetics (ESHG) Conference 2022.

## Funding

The authors are grateful to the Dutch Organisation for Health Research and Development: ZON-MW grants 912-12-109 (to B.B.A.d.V.) and Donders Junior researcher grant 2019 (B.B.A.d.V.). Part of this research was also funded by the Ministero della Salute (Current Research Funds and PNRR-MR1-2022-12376811, to M.A.T.).

## Author Contributions

Conceptualization: P.J.v.d.S., S.M., G.W.E.S.; Data Curation: P.J.v.d.S., S.M., G.W.E.S.; Formal Analysis: P.J.v.d.S., A.J.M.D., M.A.L., G.W.E.S.; Funding Acquisition: P.J.v.d.S., M.T., B.B.A.d.V., G.W.E.S., Investigation: P.J.v.d.S., A.J.M.D., M.A.L., G.W.E.S.; Methodology: P.J.v.d.S., A.J.M.D., M.A.L., B.B.A.d.V., B.S., M.A., G.W.E.S.; Project administration: P.J.v.d.S., S.M., A.J.M.D., M.A.L., B.B.A.d.V., B.S., M.A., G.W.E.S.; Resources: P.J.v.d.S., S.M., A.J.M.D., D.W., M.A.L., K.M.B., C.A., M.M.B., C.B., A.B., C.C., T.D., M.A.L.D., K.D., L.F., M.M.v.H., K.J., M.J.K., J.K., M.K., K.L., N.M., H.M., M.A.M., D.M., M.N., C.N., A.N., V.O., O.P., J.A.R., R.R., F.G.R., J.A.R., R.S., S.A.S., E.S.A., D.S., M.T., M.A.T., S.T., N.W., J.W., B.B.A.d.V., B.S., M.A., G.W.E.S.; Software: P.J.v.d.S., A.J.M.D., M.A.L., B.B.A.d.V., B.S., M.A., G.W.E.S.; Supervision: G.W.E.S.; Validation: P.J.v.d.S., A.J.M.D., M.A.L., B.B.A.d.V., B.S., M.A., G.W.E.S.; Visualization: P.J.v.d.S., M.A.L., G.W.E.S.; Writing: P.J.v.d.S., G.W.E.S.; Writing - review & editing: P.J.v.d.S., S.M., A.J.M.D., D.W., M.A.L., K.M.B., C.A., M.M.B., C.B., A.B., C.C., T.D., M.A.L.D., K.D., L.F., M.M.v.H., K.J., M.J.K., J.K., M.K., K.L., N.M., H.M., M.A.M., D.M., M.N., C.N., A.N., V.O., O.P., J.A.R., R.R., F.G.R., J.A.R., R.S., S.A.S., E.S.A., D.S., M.T., M.A.T., S.T., N.W., J.W., B.B.A.d.V., B.S., M.A., G.W.E.S.
## Ethics Declaration

The Institutional Review Board of Leiden University Medical Center, Leiden, The Netherlands provided approval waivers (no: P17.301 and G21.129). Clinical data were deidentified by allocating patient numbers. Where possible, informed consent was obtained by the referring clinician.

Written consent was obtained and archived for all included patient photos.

## Conflict of Interest

Bekim Sadikovic is a shareholder in EpiSign Inc. All other authors declare no conflicts of interest.

## Additional Information

The online version of this article (https://doi.org/10.1016/j.gim.2024.101283) contains supplemental material, which is available to authorized users.

## Affiliations

¹Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands; ²CPDPN, Pôle mère enfant, Maison de Santé Protestante Bordeaux Bagatelle, Talence, France; ³Centre de Référence Anomalies du Développement et Syndromes Malformatifs, FHU TRANSLAD, CHU Dijon, Dijon, France; ⁴Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands; ⁵Institute of Medical Genetics, Kepler University Hospital Med Campus IV, Johannes Kepler University, Linz, Austria; ⁶Verspeeten Clinical Genome Centre, London Health Sciences Centre, London, ON, Canada; ⁷Children's Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON, Canada; ⁸Department of Genetics, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada; ⁹Sección Genética Médica, Hospital de Niños Dr. Ricardo Gutiérrez, Buenos Aires, Argentina; ¹⁰Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy; ¹¹Medical Genetics, University of Siena, Siena, Italy; ¹²Service de Génétique Médicale, Centre Hospitalier Universitaire de Nantes, Nantes, France; ¹³Centre Hospitalier Universitaire de Nantes, UF de Foeto-pathologie et Génétique, Nantes, France; ¹⁴Service de Génétique Médicale, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France; ¹⁵Department of Medical Sciences, University of Torino, Torino, Italy; ¹⁶Service de Génétique, Génomique, et Procréation, Centre Hospitalier Universitaire Grenoble Alpes, La Tronche, France; ¹⁷Inserm, CNRS UMR 5309, Institut pour l'Avancée des Biosciences (IAB), Université Grenoble Alpes, Grenoble, France; ¹⁸Department of Genetic Medicine, Belfast City

Hospital, Belfast, Northern Ireland, United Kingdom; ¹⁹Medical Genetics, IRCCS Bambino Gesù Children Hospital, Rome, Italy; ²⁰Center for Human Genetics, University Hospitals Leuven, Leuven, Belgium; ²¹Genetics of Developmental Disorders, Inserm - Bourgogne Franche-Comté University, UMR 1231 GAD Team, Dijon, France; ²²Section Clinical Genetics, Department of Human Genetics, Amsterdam University Medical Centers, Amsterdam, The Netherlands; ²³Amsterdam Reproduction and Development, Amsterdam University Medical Centers, Amsterdam, The Netherlands; ²⁴Department of Pediatrics, Université de Montréal, Montreal, QC, Canada; ²⁵Centre de Recherche et Centre Hospitalier Universitaire Sainte-Justine, Montreal, QC, Canada; ²⁶Wessex Clinical Genetics Service, University Hospital Southampton, Princess Anne Hospital, Southampton, United Kingdom; ²⁷Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; ²⁸Laboratoire de Génétique Chromosomique et Moléculaire, Pôle de Biologie, CHU de Dijon, Dijon, France; ²⁹Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada; ³⁰Center for Clinical Genetics, Sydney Children's Hospital, Randwick, New South Wales, Australia; ³¹University of New South Wales, School of Women's and Children's Health, Faculty of Medicine and Health, Kensington, New South Wales, Australia; ³²Molecular Genetics and Functional Genomics, Bambino Gesù Children Hospital, IRCCS, Rome, Italy; ³³Translational Cytogenomics, IRCCS Bambino Gesù Children Hospital, Rome, Italy; ³⁴Department of Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom; ³⁵Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands; ³⁶Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; ³⁷Baylor Genetics Laboratories, Houston, TX; ³⁸School of Women's and Children's Health, UNSW Medicine and Health, University of New South Wales, Sydney, New South Wales, Australia; ³⁹Sydney Children's Hospitals Network, Westmead, New South Wales, Australia; ⁴⁰Children's Hospital Westmead Clinical School, University of Sydney, Sydney, New South Wales, Australia; ⁴¹Department of Endocrinology and Genetics, University Clinic for Children's Diseases, Medical Faculty, University Sv. Kiril I Metodij, Skopje, Republic of North Macedonia; ⁴²Department of Rehabilitation Medicine, Maastricht University Medical Center/Adelante, Rehabilitation, Maastricht, The Netherlands; ⁴³Greenwood Genetic Center, Greenwood, SC; ⁴⁴Molecular Biotechnology Center "Guido Tarone," University of Turin, Turin, Italy; ⁴⁵Service de Gynécologie-Obstétrique, Centre Hospitalier Universitaire de Nantes, Nantes, France; ⁴⁶Nun, INRAE, Physiol, UMR1280 AN, Université de Nantes, Nantes, France; ⁴⁷Department of Genetics, Valley Children's Hospital, Madera, CA; ⁴⁸Stanford University, Palo Alto, CA; ⁴⁹Department of Human Genetics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
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