Identification of a Novel SHANK2 Pathogenic Variant in a Patient with a Neurodevelopmental Disorder

Genetic defects in the SHANK2 gene, encoding for synaptic scaffolding protein, are associated with a variety of neurodevelopmental conditions, including autism spectrum disorders and mild to moderate intellectual disability. Until now, limited patient clinical descriptions have been published. Only 13 unrelated patients with SHANK2 pathogenic variations or microdeletions have been reported worldwide. By Exome Sequencing, we identified a de novo stop-gain variant, c.334C>T, p.(Gln112*), in an Italian patient with a neurodevelopmental disorder. The patient (9 years old) presented the following facial features: a flat profile, thick eyebrows, long eyelashes, a bulbous nasal tip and a prominent columella, retracted ears, dental anomalies. The patient showed speech delay and mild neuromotor delay but not autism spectrum disorder. In conclusion, this patient with a novel pathogenic variant in SHANK2 enlarges the phenotypic spectrum of SHANK2-mutated patients and demonstrates that the severity of SHANK2-associated disorders is highly variable.


Introduction
Neurodevelopmental disorders (NDDs) are a group of heterogeneous conditions affecting 2-5% of children and include autism spectrum disorder (ASD), intellectual disability (ID), developmental delay (DD), and epilepsy [1]. Different phenotypes often coexist in the same patient, thus making classification difficult. The genetic etiology underlying NDDs is highly heterogeneous, with varying degrees of genetic overlap and penetrance or expressivity across the phenotypes [2 -10].
Genes of the SHANK (SH3 and multiple ankyrin repeat domains protein) family (comprising SHANK1, SHANK2 and SHANK3) have been linked to a spectrum of neurodevelopmental disorders. The SHANK2 gene encodes for a postsynaptic scaffolding protein at glutamatergic synapses in the brain, essential for proper synapse formation, development and plasticity [11,12]. By genome-wide microarray analysis for copy number variants (CNVs) in a German cohort of 184 unrelated individuals with ID and a series of 396 Canadian individuals with ASD, Berkel et al. found one patient in each cohort who had a de novo deletion in the SHANK2 gene [13]. In the SFARI gene database, SHANK2 is categorized as a high-confidence autism risk gene. Shank2 mutant mice exhibit ASD-like behavioral deficits and synaptic dysfunctions [14]. To date, only 13 SHANK2 mutated patients Genes 2022, 13, 688 2 of 8 have been described worldwide, and thus the phenotypic spectrum of the condition is only partially described [15][16][17][18][19][20][21][22][23][24]. Among these patients, all individuals carried de novo variants, six variants resulting in a premature stop codon, and five microdeletions encompassing the gene. All patients had mild to moderate ID, and autism was reported in nearly all of them. There are also reported cases with rare missense SHANK2 variants inherited from unaffected parents, suggesting the existence of low-penetrance alleles [13].
In the present study, we performed clinical exome sequencing in a patient with a neurodevelopmental disorder and found a de novo SHANK2 pathogenic variant, c.334C>T, p.(Gln112*). Interestingly, the patient showed mild intellectual disability and speech delay, without any signs of ASD, expanding the phenotypic spectrum associated with SHANK2 mutations.

Sample and DNA Extraction
Peripheral blood samples were collected in EDTA tubes from the proband and her parents at the Medical Genetics Unit of the Azienda Ospedaliera Universitaria Senese (A.O.U.S, Siena, Italy). Following written informed consent for both diagnostic and research purposes, genomic DNA samples were extracted using MagCore HF16 (Diatech Lab Line, Jesi, Ancona, Italy).

Exome Sequencing
Exome sequencing was performed on genomic DNA samples of the proband and both parents.
Library preparation and exome capture were obtained using the Illumina Nextera Flex for Enrichment KIT according to the manufacture's protocol, and sequencing was performed using the Illumina NovaSeq 6000 platform (Illumina San Diego, CA, USA). Alignment of raw paired-end reads to the reference genome (version hg19) was performed with BWA Enrichment (v2.1.2). Variant calling was obtained using the Genome Analysis ToolKit (GATK).
Finally, for the interpretation of the variants, the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines were used [25].

Sanger Sequencing
To confirm the SHANK2 variant, we performed Sanger sequencing of exon 3, including the flanking intron sequences of the gene (NM_133266.3) in the proband and her parents. DNA was amplified by PCR using specific primer pairs. Subsequently, the purified PCR products were applied to Sanger sequencing to affirm the mutation, using an ABI PRISM3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The Sanger sequencing results were analyzed with Sequencher software V.4.9 (Gene Codes, Ann Arbor, MI, USA).

Mutation Nomenclature
The mutation is described according to Human Genome Variations Society (HGVS). Nucleotide numbers are derived from the cDNA sequence of SHANK2 (GenBank accession NM_133266.3).

Clinical Description
The patient was a 9-year-old female, the first child of healthy parents. She was born from an uncomplicated pregnancy at full term. Family history was negative for ASD or other neurodevelopmental disorders. The auxological parameters at birth were: length 51 cm (71 • percentile) and weight 3450 g (25 • -50 • percentile).
The mother reported that the baby showed hypovalid suction. She described the onset of crawling at 12 months, autonomous walking at 15 months, production of single words at 2 years and 6 months and production of simple sentences at 6 years. Sphincter control was acquired at about 3 years. Eye contact with parents has always been good. At the age of about 2 years and 6 months, the girl began psychomotor therapy and speech therapy, with significant benefits. The girl has support at school and shows considerable difficulties in mathematical calculation. Good interaction with peers is reported. Her diet is varied and her sleep-wake rhythm is normal. She underwent audiometric, ophthalmological, brain MRI and EEG evaluation, all of which were normal.
including the flanking intron sequences of the gene (NM_133266.3) in the proband and her parents. DNA was amplified by PCR using specific primer pairs. Subsequently, the purified PCR products were applied to Sanger sequencing to affirm the mutation, using an ABI PRISM3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The Sanger sequencing results were analyzed with Sequencher software V.4.9 (Gene Codes, Ann Arbor, MI, USA).

Mutation Nomenclature
The mutation is described according to Human Genome Variations Society (HGVS). Nucleotide numbers are derived from the cDNA sequence of SHANK2 (GenBank accession NM_133266.3).

Clinical Description
The patient was a 9-year-old female, the first child of healthy parents. She was born from an uncomplicated pregnancy at full term. Family history was negative for ASD or other neurodevelopmental disorders. The auxological parameters at birth were: length 51 cm (71° percentile) and weight 3450 g (25°-50° percentile).
The mother reported that the baby showed hypovalid suction. She described the onset of crawling at 12 months, autonomous walking at 15 months, production of single words at 2 years and 6 months and production of simple sentences at 6 years. Sphincter control was acquired at about 3 years. Eye contact with parents has always been good. At the age of about 2 years and 6 months, the girl began psychomotor therapy and speech therapy, with significant benefits. The girl has support at school and shows considerable difficulties in mathematical calculation. Good interaction with peers is reported. Her diet is varied and her sleep-wake rhythm is normal. She underwent audiometric, ophthalmological, brain MRI and EEG evaluation, all of which were normal.

Genetic Analysis
Exome Sequencing analysis was performed on the patient and her parents. We obtained a mean depth of coverage of 80X. A total of 11.670 genetic variants on average for each sample was yielded.

Genetic Analysis
Exome Sequencing analysis was performed on the patient and her parents. We obtained a mean depth of coverage of 80X. A total of 11.670 genetic variants on average for each sample was yielded.
The analysis by classification filtering revealed a de novo stop-gain variant c.334C>T, p.(Gln112*) in exon 3 of the SHANK2 gene on chromosome 11 (Figure 2).

Discussion
In the present study, we report the identification of one rare patient with a SHANK2 pathogenic variant, c.334C>T, p.(Gln112*), showing a neurodevelopmental disorder. To date, only 13 cases with de novo SHANK2 alterations have been reported worldwide, and the resulting phenotypes are poorly described (Table 1) [15][16][17][18][19][20][21][22][23][24]. Sanger sequencing confirmed the heterozygous variant in the proband and not in parents ( Figure 3). This frameshift deletion has not been previously reported and it is absent in the ExAC and gnomAD databases. The variant has been submitted to the LOVD database (https://www.LOVD.nl, accessed on 10 February 2022) with the following ID: 0000836786.
obtained a mean depth of coverage of 80X. A total of 11.670 genetic variants on average for each sample was yielded.
The analysis by classification filtering revealed a de novo stop-gain variant c.334C>T, p.(Gln112*) in exon 3 of the SHANK2 gene on chromosome 11 (Figure 2). Sanger sequencing confirmed the heterozygous variant in the proband and not in parents ( Figure 3). This frameshift deletion has not been previously reported and it is absent in the ExAC and gnomAD databases. The variant has been submitted to the LOVD database (https://www.LOVD.nl, accessed on 10 February 2022) with the following ID: 0000836786.
All reported patients had ID, ranging from mild to moderate [15][16][17][18][19][20][21][22][23][24]. In accordance, the girl described here showed mild intellectual disability. Nearly all previous patients showed autism, while our case does not show any sign of ASD. Our patient manifested language delay, and this represents a key feature of the condition being present in all reported patients [15][16][17][18][19][20][21][22][23][24]. Additional neurologic features such as anxiety or repetitive behavior were frequently observed but were absent in the present case [13,15,16,[21][22][23][24]. The delineation of a common facial phenotype is hampered by the lack of facial descriptions in previous papers. Here, the patient shows a flat profile, thick eyebrows, long eyelashes, a nose with a bulbous tip and a prominent columella, large and spaced teeth, retracted ears. This variant, c.334C>T, p.(Gln112*), in SHANK2 is an early truncating variant with abolishment of the major domains of the protein (SH3, PDZ domain, Proline-rich region, SAM). In accordance with the present case, SHANK2 truncating variants are all de novo events in affected individuals. Only SHANK2 missense variants found in patients with NDD are inherited from healthy parents and probably represent low-penetrance alleles [13]. Dhaliwal et al. identified a missense variant in the SHANK2 gene in association with multiple inherited variants in other ASD-associated genes (RELN, SHANK2, DLG1, SCN10A, KMT2C and ASH1L) in a family with three affected children and hypothesized that additional genetic/epigenetic factors together might be necessary to develop ASD [26]. Moreover recent studies support that genetic defects in the SHANK2 gene are implicated in neuropsychiatric disorders [27][28][29]. The mutation type as well as the presence of modifiers in the genome can be fundamental. Leblond and colleagues recently identified CNVs as putative modifiers of the phenotype in accordance with a "multiple-hit model" [18]. Additional studies are necessary to delineate more robust genotype-phenotype correlations.
In Shank2 mutant mouse models, anxiety-like behavior, hyperactivity, abnormal social behavior and dysregulation of synaptic molecules have been observed in specific regions of the brain [30].
A possible pathogenic mechanism has been elucidated by the use of human induced pluripotent stem cells (hiPSCs). Lutz et al. generated hiPSCs from a patient carrying a heterozygous deletion of SHANK2 and from the unaffected parents. They observed a reduction in growth cone size and a transient increase in neuronal soma size during neuronal maturation in patients' derived cells. Furthermore, the extracellular signal-regulated kinase (ERK) pathway resulted dysregulated [31]. Another hiPSC study showed that human neurons with ASD-associated haploinsufficient SHANK2 mutations had increased synaptic connectivity relative to controls [32]. These findings reinforce the hypothesis of SHANK2 involvement in neurodevelopmental disorders. In addition, the generation of animal models and hiPSC studies are important to discover and design therapeutic strategies.
Regarding the treatment of ASD, the supplementation of zinc (Zn) is a possible preventive strategy. Zinc is a metal involved in the developmental and functioning of the central nervous system (CNS) and is crucial for normal cognitive functions and emotional behaviors. Zn is also responsible for maintaining viable synapses by stabilizing SHANK2/3 postsynaptic scaffolding proteins [33,34]. Zn deficiency is a risk factor that may contribute to the pathophysiology of neurodevelopmental disorders such as ASD. Zinc supplements improved the behavioral deficits in animal models of ASD and in Shank3-mutated animals [35]. Clinical trials are still needed to validate the beneficial therapeutic effects of zinc supplements in ASD patients.
In conclusion, our findings underline that ASD is not necessarily a constant clinical feature in SHANK2-mutated patients and, in accordance with previous literature, indicate language impairment as a major feature in these patients. A peculiar facial phenotype may be observed, but additional studies with comprehensive clinical descriptions are required.  Institutional Review Board Statement: Ethical review and approval were not required for the study on human participants in accordance with local legislation and institutional requirements.

Informed Consent Statement:
The parents gave informed consent for diagnostic testing and research studies.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.