Extended phenotypic characterization of a novel Helsmoortel‐van der Aa syndrome case series

The neurodevelopmental disorder known as Helsmoortel‐van der Aa syndrome (HVDAS, MIM#616580) or ADNP syndrome (Orphanet, ORPHA:404448) is a multiple congenital anomaly (MCA) condition, reported as a syndrome in 2014, associated with deleterious variants in the ADNP gene (activity‐dependent neuroprotective protein; MIM*611386) in several children. First reported in the turn of the century, ADNP is a protein with crucial functions for the normal development of the central nervous system and with pleiotropic effects, explaining the multisystemic character of the syndrome. Affected individuals present with striking facial dysmorphic features and variable congenital defects. Herein, we describe a novel case series of HVDAS Italian patients, illustrating their clinical findings and the related genotype–phenotype correlations. Interestingly, the cutaneous manifestations are also extensively expanded, giving an important contribution to the clinical characterization of the condition, and highlighting the relation between skin abnormalities and ADNP defects.

HVDAS syndrome is characterized by neurodevelopmental delay (intellectual disability, autism spectrum disorder, language development, behavioral disturbances) and distinctive craniofacial abnormalities, mostly including long palpebral fissures, ptosis, deeply grooved tongue, broad nose, and down-turned mouth.Visual and ophthalmologic impairment, extremities anomalies, and visceral defects (congenital cardiopathy, urogenital malformations, gastrointestinal disease) can be also a part of the clinical picture.The original description of ADNP pathogenic variants affecting development and intellectual abilities was in 2007 by Borodozin et al. and modeled in mice by Gozes and colleagues (Borozdin et al., 2007;Vulih-Shultzman et al., 2007).
In the present research, we have analyzed the clinical findings of a novel Italian case series, broadening the associated phenotype.
Notably, we also considered the skin findings in the clinical delineation of the present studied cohort, providing novel clinical observations.The molecular diagnosis was obtained for all patients through exome sequencing analysis.

| RESULTS
We summarized the main clinical features of the present cohort in Table 1, while all phenotypic characteristics are detailed in the Table S1 as follows: 60% of patients were females, 40% were males.
Dysmorphic traits were identifiable in 100%: anomalous eyelids were present in 100% (downslanted in 60%; short and downslanted in 20%), high anterior hairline in 67%; ptosis and hypertelorism were appreciable in two patients (Figure 1).Considering the oral region, all patients showed anomalies of the philtrum, which appeared long and smooth in 47%, long in 27%, as well as short; tooth disorders were diagnosed in 89% (macrodontia of the permanent upper central incisors in 67%, diastema in 55% and precocious dentition in 22%); everted lower lip vermillion was detected in the entire cohort while thin upper lip vermillion in 80% (Figure 1).The nose had a peculiar morphology in 100%, including broad nasal tip (60%), depressed nasal bridge (53%), depressed and wide nasal bridge (20%), wide nasal bridge and low hanging columella (each in 13%) (Figure 1).Defects of the external ear, that were described in a previous wide case series as small or dysplastic, low-set, and posteriorly rotated All the patients manifested visual/ophthalmologic issues in particular: exophoria (36%), astigmatism, hypermetropia (each in 27%), strabismus (18%), corneal opacity, lost fixation, pseudopapillary halo, asymmetry of fundus oculi, and atrophy/hypoplasia of anterior visual pathways (singly in 9%).

| SKIN PHENOTYPE
We illustrate the skin manifestations in a distinct paragraph, considering the novelty of the research field.Dermatological anomalies have been reported by parents in 89% of our cohort, F I G U R E 1 Facial features of the present cohort of patients.Patient number is indicated according to the Table S1.

| MOLECULAR DATA AND GENOTYPE-PHENOTYPE CORRELATIONS
In the present cohort, the most recurrent variant was the p.Tyr719* (c.2157C>G and c.2157delC), which was disclosed in six patients; the c.2496_2499del(p.Asn832Lysfs*81) and c.2188C>T(p.Arg730*) variants were respectively identified in three and two patients (Table S1).S1).The last two fall within one zinc finger domain of the protein (Zamostiano et al., 2001) (Figure 3a).
All variants are de novo and predicted to result in protein truncation, including five frameshift and three nonsense changes.
In the present cohort, no specific genotype-phenotype correlations have been identified for the following features: DD/ASD, craniofacial dysmorphisms, and extremities anomalies, which are present in all patients, regardless of the disclosed variant (Figure 3b).S1.
In this regard, we found the ADNP-type 2 syndrome in 10 patients and the ADNP-type 1 in the remaining 5 (Table S1).According to previous data, we found recurrent infections (respiratory) and gastrointestinal involvement mostly in ADNP-type 2 syndrome patients (Table S1).

| DISCUSSION
We have analyzed the full phenotype of a novel Helsmoortel-van der Aa, case series, the first from Italy.
Our clinical data agree with what is known about the condition, also respect to the methylation signatures, as illustrated above.
In terms of phenotype/genotype correlations, genome-edited mice and cells mimicking the p.Tyr719* human pathogenic variant (Tyr mouse), suggested a gain of toxic function, compared to haploinsufficiency or a mutation mimicking the p.Ser404* in humans (Ganaiem et al., 2022;Karmon et al., 2022).Indeed, the Tyr mouse displayed more extensive motor impairments compared to the haploinsufficient mice (Bend et al., 2019), while in cell culture, the p.Tyrlike human pathogenic variant resulted in a more severe phenotype (reduced cell numbers) compared to the p.Ser404*-like human pathogenic variant.As mentioned above, other studies suggested two episignatures on ADNP, Epi-ADNP-1 pathogenic variants occupying the N-and C-terminus, and epi-ADNP-2 pathogenic variants centered on the nuclear localization signal (Bend et al., 2019).Furthermore, some frame-shift pathogenic variants may include a "protective" sequence addition (Breen et al., 2020;Gozes & Shazman, 2023).Together, these suggest a strong ADNP pathogenic phenotype, regardless of the mutation, coupled with genotype/phenotype interactions, which may be masked, in part, due to the crucial roles of ADNP and the individual background genetics as well as environmental conditions (Bend et al., 2019).
Interestingly, most of our patients display macrodontia of the permanent upper central incisors, which is not a cardinal feature in the HVDAS and is reputed as a distinctive sign of KBG syndrome (KBGS, MIM#148050), a rare chromatin disorder associated with variants in ankyrin repeat-containing cofactor 1 gene (ANKRD11, MIM*148050) (Sirmaci et al., 2011).On the other hand, distinctive facies reminiscent of Rubinstein-Taybi syndrome 1 (RTS1, MIM#180849) and associated with broad halluces have been recognized in patient 10 (Figures 1 and   2).Taken together, these observations demonstrate the high clinical connection between the several syndromes of the epigenetic machinery, comprising HVDAS.
Regarding morphology of the extremities, patient 15 exhibits a peculiar and apparently novel foot malformation pattern, resembling a split foot (Figure 2), which needs to be further investigated by extensive radiography.Mechanistically, ADNP contains a homeobox domain (Zamostiano et al., 2001) and has been shown to be critical to neural tube closure (Pinhasov et al., 2003), partially explaining, the dysmorphism associated with pathogenic mutations as observed and detailed here and also detected during pregnancy (Rosenblum et al., 2023).
CHD, neuropsychiatric anomalies, gastrointestinal issues, sleep disorders, respiratory disease, and genitourinary malformations were registered as comorbidities in 25%-50% (Table 1; Table S1).Of note, patient 15 shows a unique congenital defect impairing the anus and external genitalia, which could suggest a development inborn error of the embryo cloaca structures (Table S1).Interestingly, a rare congenital anomaly, comprising an imperforate anus, was reported before (Gozes, Patterson, et al., 2017).
Skin manifestations were discovered to be an important clinical finding in this cohort (89%) (Table 1; Table S1).A single paper analyzing the skin phenotype in a sporadic patient with a thin dermis, hyperkeratotic lesions, and delayed wound healing has been previously provided (Mollinedo et al., 2019).This work demonstrated through in vitro and in vivo studies an ADNP involvement in skin health, which is impaired in mutants (Mollinedo et al., 2019).Furthermore, based on information from 23 ADNP families of a previous study, the authors reported that some subjects with the p.Tyr719* variant display skin alterations, including delicate skin, slow wound healing, eczema, and rashes (Mollinedo et al., 2019).All these aspects point out the common embryonic origin of the brain, tooth enamel, and epidermis, as already postulated (Mollinedo et al., 2019).Thus, Helsmoortel-van der Aa syndrome with ADNP linked to SWI/SNF or BAF chromatin remodeling complex (Mandel & Gozes, 2007) manifests a skin involvement, like other chromatin disorders, including Koolen-de Vries (KDVS, MIM#610443) and BAF complex-related syndromes (Khazanchi et al., 2019;Koolen et al., 2016;Zapata-Aldana et al., 2023;Zollino et al., 2015).
A potential limit of our study can be represented by the relatively young age of patients, which is common with the prior case series (Dingemans et al., 2023), and related to the recent detection of the molecular cause of the condition, probably underestimated in the past.Another limitation could be the impossibility of directly evaluating all patients, living in different cities and often with poor tolerance to long travels, which is easily understandable if we think of the neurocognitive involvement.However, our goal will be the clinical assessment of each patient soon.
In conclusion, our genotype-phenotype evidence expands Helsmoortel-van der Aa syndrome phenotype, highlighting skin findings, a previously poorly investigated feature, and offering novel future therapeutic targets.Clinicians have long envisioned a time when artificial intelligence could assist with difficult diagnoses.Further specific features of HVDAS including skin abnormalities might contribute to distinguishing among syndromes with other similar features.Additional studies and the indispensable support of patients' families will be crucial for a better phenotype definition, disease management, and targeted therapeutics.
This study was performed according to the Helsinki Declaration and approved by the IRB of the main involved Institution (ID#578/3, 2023).All families authorized the processing of personal data and publication of clinical images.A total of 15 patients, mostly pediatrics, representing the entirety of individuals of the ADNP Syndrome Italian Association when this manuscript was in preparation, have been considered in this study.Patients' information and clinical images have been obtained from the medical database of the Association, a recently founded family group, sharing clinical and scientific updates regarding the disorder.The private database of the Association was conceptualized and managed by the first author (GP), who is a clinical geneticist experienced in dysmorphology and a member of the scientific board of the Association, along with the last author (IG), the discoverer of ADNP.Clinical features were reviewed in a panel of patients' photographs.All individuals were enrolled for the first time, except patient 1, which has been already published by Pascolini et al. (2018; patient 1) and Breen et al. (2020; patient 25R).

F
I G U R E 2 Extremities morphology of the studied patients.Patient number is indicated according to the Table

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I G U R E 3 (a) ADNP protein structure with its functional domains.The novel variants are depicted above, and known changes are below.Variants are differently colored.(b) Schematic table resuming the main clinical features of the present cohort, which are reported for every single patient and colored according to the disclosed variant.For genotype-phenotype correlation details see the text.
Main clinical findings of the present HVDAS cohort.
(Van Dijck et al., 2019)et al., 2019), were not evaluable in this study, due to the lack of suitable images.