**4. Discussion**

We described the clinical phenotype of a cohort of eight patients that were highly suggestive of 22q11.2DS without harboring genomic aberrations of chromosome 22. The cardinal features of 22q11.2DS, such as congenital heart defects, hypoparathyroidism, facial dysmorphisms, and immunological abnormalities, were observed in this cohort of patients.

The typical cardiac defects, such as tetralogy of Fallot, right-sided aortic arch, and truncus arteriosus, were identified in a few patients of our cohort, whereas minor cardiac anomalies were observed with higher frequency and uncommon defects, such as aortic coarctation and hypoplastic left heart syndrome, were also described. Peculiar facial dysmorphic features, such as hypertelorism, narrow palpebral fissures, epicanthal folds, and micrognathia, were observed in all the patients of the study; furthermore, some uncommon traits, such as wide ear pad, low set ears, flat nasal bridge, and enlarged nasal, were also found. Hypoparathyroidism was not associated with cardiac defects, contrary to what is reported in 22q11.2DS. Concerning the neurodevelopmental disorders, they did not differ from 22q11.2DS, with speech and psychomotor delays being the most frequent manifestations identified during the follow-up. The frequency of the other phenotypic features was similar to that of typical 22q11.2DS [2,4,10,30].

The immunological profile did not substantially differ from typical 22q11.2DS, showing various degrees of mild or moderate immunodeficiency, mainly related to the cellmediated compartment. We reported a reduced thymic output entailing low RTEs and reduced numbers of both CD4+ and CD8<sup>+</sup> naïve T lymphocytes. The decline in naïve and the increase in memory T-cell populations observed in our cohort could have been due not only to the impaired thymic output but also to the accelerated conversion of naïve to memory phenotype, secondary to multiple mechanisms, such as infectious exposures or homeostatic expansion, as we previously reported [31]. No severe phenotype resembling a leaky SCID was observed in our cohort. Thymic hypoplasia and, more recently, the immature status of the thymus (mainly referred to as an impairment of the epithelium function) described in the majority of cases of 22q11.2DS [5,32] may have underlain the immunological abnormalities observed in our population. We previously described a defective Tregs number in 22q11.2DS [23] that, when associated with an impaired expression of AIRE-dependent tissue-restricted antigens, leads to an impaired peripheral tolerance and consequent escape of autoreactive T cells [32]. Conversely, the Tregs number was normal in our patients, as well as the number of switched memory B cells, which were reported (together with a low level of naïve T helper cells) as strong predictors for the development of autoimmune disorders in DGs patients, particularly in their adult life [33]. Interestingly, a low level of DCs was observed in half of our patients, as we recently reported in 22q11.2DS [23], most prevalently in the mDC compartment. We argue that this

finding may contribute to the observed high susceptibility toward developing infectious diseases and autoimmune manifestations of these patients. Concerning vitamin D status, the limited population analyzed did not allow for investigating its relationship with the immunological parameters, as we previously reported in 22q11.2DS. Furthermore, due to the recognized immunomodulatory role of vitamin D, it appears reasonable to suggest its supplementation also in these patients, as infections and susceptibility to autoimmune diseases represent their major concern, similarly to 22q11.2DS.

Genetic assessment in our cohort revealed six patients harboring CNVs that are never reported in normal subjects (Table 6). The genes content of these CNVs was carefully analyzed for their correlation with the phenotypic features reminiscent of 22q11.2DS.

In subject P1, a 314 kb duplication in the 2p24.1 region, where the long intergenic non-protein coding RNA 1876 (LINC1876) is harbored, was identified. According to Gene-Hancer (https://genome.ucsc.edu (accessed on 25 February 2022)), LINC1876 regulates the expression of NR4A2, a gene located distally to the duplicated region. NR4A2 encodes a steroid–thyroid hormone-retinoid receptor, acting as a nuclear receptor (NR) transcription factor, and is mainly expressed in neurons of several areas of the CNS where it is specifically required for the development and function of the neurons. The dysregulation of this gene has been associated with neurodevelopmental delay and intellectual disability with or without epilepsy [34]. Altered NR4A2 expression is thought to have caused the neurological phenotype of P1, characterized by epilepsy with psychomotor delay and cognitive impairment. It was demonstrated that the NR4A family has a role in T cell development from thymic differentiation to peripheral response against infections and cancer; the overexpression of NR4A1 and NR4A3, but not NR4A2, induces thymocyte apoptosis in vivo [35]. Although NR4A2 appears not to be involved in this mechanism, its altered expression in our patient might contribute to determining his severe deficiency of thymic output.

P4 and P5 patients were two sisters who exhibited a 365 kb duplication in the 20p11.22 region, including the PAX1 gene. PAX1 is a member of the paired box (PAX) family of transcription factors that plays a critical role in human embryogenesis at the level of pharyngeal pouches, involving the development of the thymus, tonsils, parathyroid glands, thyroid, and middle ear [26,36,37]. PAX1 homozygous loss-of-function variants are causative of otofaciocervical syndrome 2 with T-cell deficiency (OTFCS2) (#615560 OMIM), which may include a severe combined immunodeficiency (SCID) due to abnormal thymic epithelium development [26]. The main aspects of this syndrome are facial anomalies, cup-shaped low-set ears, preauricular fistulas, hearing loss, branchial defects, skeletal anomalies, and mild intellectual disability [24,25]. Although the effects of PAX1 duplication are still unknown, it might alter the embryonic development of the pharyngeal region. It is possible to hypothesize that PAX1 dosage alteration can contribute specifically to the otolaryngological manifestations, such as the hearing loss observed in patient P5 and to the dysmorphic auricular appendix in patient P4, as well to the hypoparathyroidism and immunological alterations found in both sisters, which is usually observed in 22.11.2DS.

In patient P6, the coexistence of two CNVs, located on 11p15.5 and the pseudoautosomal regions Xp22.33/Yp11.32 was detected. The 11p15.5 duplication was rich in genes, five of which are related to known syndromes with an autosomal recessive inheritance (#617637, #606003, #609304, #610717, #609057 OMIM). The effects of their duplication are unknown, but, according to their expression and function, none of them seem to influence the patient's phenotype.

The duplication in the pseudoautosomal region Xp22.33/Yp11.32 included SHOX, encoding a homeodomain transcription factor involved in cell cycle and growth regulation. SHOX deletions cause well-defined pathologic phenotypes, mainly including short stature and skeletal abnormalities (#249700, #127300, #300582 OMIM). Recently, it was highlighted that microdeletions encompassing this gene are a risk factor for autism spectrum disorders and neurodevelopmental defects [27]. We outline the possible role of this variant in determining the speech delay observed in the patient. In the human embryo, SHOX is

expressed both in the limbs and in the first and second pharyngeal arches, from which originate the maxilla, mandible, and several bony elements of the external and middle ear [38]. It could be speculated that conductive hearing loss may be attributed to the dysregulation of this gene.

In P7, a 17q21.13 deletion was identified, which is causative of the Koolen–De Vries syndrome (KDVS #610443). KDVS has variable expressivity and a wide clinical spectrum that can overlap with DGS. The pathologic features of the patient include neurodevelopmental delay, anxiety disorder with psychotic signs, as well as facial dysmorphisms (long face, malar flatness, hooded eyelids resulting in the appearance of narrow palpebral fissures, and bulbous nasal tip), which overlap between both syndromes [39]. The other clinical features, such as skeletal anomalies and otolaryngological manifestations, are less common in KDVS. Interestingly, a patient with KDVS was also identified in a previously reported cohort of DGS without a 22q11.2 deletion [8]. The overlapping phenotypes of these two syndromes could be attributed to an underlying common genetic pathway. The master gene of KDVS is KANSL1, a protein-coding gene that belongs to a histone acetyltransferase (HAT) complex. Even if direct interactions between KANSL1 and the protein-coding genes located in the 22q11.2-deleted region have not been demonstrated, a common miRNA regulatory network has been identified. miRNAs play a role in 22q11.2DS [40]; interestingly, it was highlighted that specific miRNAs (such as miR-106b-5p, miR-148a-3p, miR-23b-3p, miR-17-5p, miR-149-5p, and miR-130b-3p) involved in the KANSL1 regulation also regulate DGCR14, DGCR2, TXNRD2, MRPL40, and CRKL genes, which are included in the 22q11.2-deleted region [41].

Patient P8 showed two deletions: the first in 17p13.2 harboring the WSCD1 gene and the second one in Xq24 encompassing the CXorf56 and UBE2A genes. The clinical effects of WSCD1 haploinsufficiency are unknown, whereas the deletion of CXorf56 and UBE2A was related to "Intellectual developmental disorder, X-linked 107" (#301013) and to "UBE2A deficiency syndrome" or "X-linked Nascimento-type intellectual disability syndrome" (#300860), respectively. Developmental delay, motor delay, impaired speech, and mild axial hypotonia, all present in P8, could be related to this Xq24 deletion. The phenotypic spectrum of UBE2A deficiency syndrome was recently expanded, and cardiac defects, craniofacial dysmorphisms, urogenital malformations, and hypogammaglobulinemia emerged as frequent features [42,43]. Therefore, the clinical spectrum of our patient, including cardiologic and urogenital defects and facial dysmorphic features, together with hypogammaglobulinemia, could be attributed to this deletion.
