Study population

As of June 2020, 1132 families (661 in the US) consented to participate in the PMS_DN project. Of these, 754 patients had at least their demographic information entered into the registry, including 749 with phenotypic information from the clinical questionnaire (718 patients) and/or the developmental questionnaire (497 patients), and 491 individuals with genetic test results. We included in the analyses 401 patients with phenotypic information who had either 22q13 deletions with genomic coordinates defined by chromosomal microarray and encompassing SHANK3 (n = 350) or pathogenic or likely pathogenic sequence variants in SHANK3 (n = 51). As expected, there were no differences in the gender distribution of the participants (46.9% males, Table 1). Analysis of the demographic information (Table 1) revealed that the selected population was slightly younger and less ethnically diverse than the larger PMS_DN cohort. The majority of the individuals studied were White, which is likely a consequence of the socio-economic factors involved in the referral of individuals with ID and ASD for genetic testing. The 401 patients included in the analyses were distributed across countries as follows: United States 240, Spain 34, Australia 22, Canada 20, United Kingdom 19, France 15, Brazil 14, Italy 12, Belgium 3, Greece 3, Ireland 3, Chile 2, Russia 2, and one patient each from Colombia, Costa Rica, Israel, Mexico, Netherlands, New Zealand, Norway, Portugal, Romania, South Africa, Sweden, and United Arab Emirates. The completion rates of items in the clinical and developmental questionnaires ranged from 2% to 95%, with a mean completion rate of 69%. A total of 328 phenotypes, each present in at least five individuals, were included in the analyses.

Table 1 Sample characteristics and comparison with individuals excluded because of missing phenotypic or genetic dataGenetic findings

Figure 1 shows the 22q13.2-q13.33 region deleted in PMS and the genetic variants in 401 individuals included in the genotype-phenotype analysis. The variant types are summarized in Table 2. Deletion sizes ranged from 10 kb to 9.1 Mb, with a median deletion size of 3.7 Mb. Most deletions were terminal (97.1%), and were associated with translocations in 12.1% and with ring chromosome 22 in at least 8.5% of cases. The proportion of individuals with ring chromosome 22 is likely underestimated, given that the majority of individuals with deletions (57.1%) were diagnosed with chromosomal microarray and had not had a karyotype performed to exclude the presence of a ring chromosome. Thirteen individuals (3.7%) had mosaic deletions. SHANK3 sequence variants included frameshift, nonsense, splice-site and missense variants; the majority were truncating variants (Table 2). Among the individuals for whom parental testing was available, all deletions and sequence variants were confirmed to have occurred de novo.

Fig. 1

Genetic variants affecting SHANK3 in 401 PMS patients included in the study. Each line represents a patient; deletions are shown in red and SHANK3 sequence variants in blue. For simplicity, sequence variants are represented as overlapping the whole gene. SHANK3 is indicated by an arrow. Chromosomal coordinates are based on the GRCh38 genome build. Constrained genes intolerant to loss-of-function variants as measured by the LOEUF (loss-of-function observed/expected upper bound fraction) metric from gnomAD (v2.1.1) are indicated in orange (LOEUF < 0.2 darker orange, < 0.3 lighter orange; smaller LOEUF indicates higher constraint)

Table 2 SHANK3 genetic variation in 401 individuals with Phelan-McDermid syndromeGenotype-phenotype analyses

We tested 328 phenotypes in 401 patients in relation to deletion size, using the multivariate models described in the Methods section. We found that 130 phenotypes were significantly associated with larger deletions (adjusted p value < 0.05), suggesting that additional genes on chromosome 22q contribute to these clinical manifestations, independently or in association with SHANK3. Tables 3 and 4 show the phenotypes positively and negatively associated with deletion size, respectively. For complete analyses and figures of all the phenotypes, see Additional file 2 (Table S1) and Additional file 3 (Other supplementary figures).

Table 3 Phenotypes significantly and positively associated with deletion sizeaTable 4 Phenotypes significantly and negatively associated with deletion sizeaPhenotypes positively associated with deletion size

Major delays in nearly all gross motor milestones were strongly and linearly associated with larger deletion sizes (Fig. 2). These included Walks unassisted, Crawls on hands and knees, Sits when placed, Rolls over back to stomach, Climbs stairs standing up without help, Descends stairs without help, and Holds head up on his/her own (adjusted p values ranged from 9.27e−18 to 4.01e−5, ORs ranged from 2.13 to 5.31; p values and ORs for each phenotype are listed in Table 3). Large deletions were also associated with delays in fine motor skills (Deliberately releases object to a container; Transfers object between hands; Looks at, reaches and grasps distant objects; p values from 1.74e−3 to 4.87e−3, ORs from 1.58 to 1.66), coordination deficits (Difficulties maintaining balance and Coordination difficulties, p values 1.93e−9 and 1.35e−3, ORs 2.25 and 1.56, respectively), and difficulties with motor planning (p = 3.35e−2, OR 1.37). These delays and deficits were accompanied by a range of conditions related to low muscular tone, including floppy baby, neonatal hypotonia, neonatal hyporeflexia, neonatal feeding problems, fatigue, ‘weak muscles’, and poor reflexes (p values from 4.68e−10 to 1.46e−2, ORs from 1.17 to 1.39). Specific feeding difficulties in babies observed more frequently in individuals with larger deletions included Required special feeds, Swallowing problems, Difficulty latching onto the breast, Difficulty latching onto the bottle, Poor suck, and Failure to gain weight normally (p values from 3.51e−6 to 2.50e−2, ORs from 1.14 to 1.29), as well as the need for nasogastric tube (p = 1.59e−2, OR 1.33). Moreover, breathing difficulties at birth, requiring neonatal intensive care, were more common among carriers of larger deletions.

Fig. 2

Association between deletion size and age of acquisition of gross motor milestones. On the left: deletions (in red) and SHANK3 variants (asterisks), ordered by decreasing deletion size/position. The position of SHANK3 is indicated by a vertical line. On the right: status of each patient for developmental gross motor phenotypes, lined up with the respective genetic status. The shades of gray and black represent the age range at which the patients achieved gross motor milestones, with lighter shades indicating younger ages and darker shades indicating older ages. The color white indicates that the answer was either 'not applicable' or 'unsure'

Of note, all renal conditions in the clinical questionnaire, including Vesicoureteral reflux, Dysplastic kidney, Polycystic kidney (defined in the questionnaire as “multiple cysts present in one or both kidneys”, which may include multicystic dysplastic kidney), Hydronephrosis, Recurrent urinary tract infections, and Increased kidney size, were strongly associated with deletion size (p values from 2.32e−7 to 1.18e−2, ORs from 1.30 to 1.67). The results indicate that patients with renal malformations carried larger deletions (Fig. 3). Persistent fever related to recurrent urinary tract infections was also associated with deletion size (p = 7.60e−3, OR 1.53). In addition, primary lymphedema was reported more frequently in individuals with large deletions (p = 4.55e−3, OR 1.35) (Fig. 4). Individuals with large deletions also exhibited swelling of the extremities, likely reflecting the presence of lymphedema (Persistent swelling of the hands and feet and Persistent swelling of the legs; p values 2.94e−6 and 1.05e−4, ORs 1.40 and 1.39, respectively).

Fig. 3

Association between deletion size and renal abnormalities. On the left: deletions (in red) and SHANK3 variants (asterisks), ordered by decreasing deletion size/position. The positions of SHANK3 and CELSR1, recently implicated in renal defects, are indicated by vertical lines. On the right: status of each patient for renal conditions, lined up with the respective genetic status. The color black indicates the presence of the phenotype, gray indicates the absence of the phenotype, and white indicates missing information

Fig. 4

Association between deletion size and lymphatic-related conditions. On the left: deletions (in red) and SHANK3 variants (asterisks), ordered by decreasing deletion size/position. The positions of SHANK3 and CELSR1, recently implicated in lymphedema, are indicated by vertical lines. On the right: status of each patient for lymphatic-related conditions, lined up with the respective genetic status. The color black indicates the presence of the phenotype, gray indicates the absence of the phenotype, and white indicates missing information

In terms of cardiac abnormalities, the presence of a heart murmur (p = 3.43e−5, OR 1.32) and congenital heart defects (p = 4.91e−3, OR 1.25) were associated with larger deletions, including patent ductus arteriosus (n = 15), ventricular septal defect (n = 10), ‘hole in the heart’ (n = 9), bicuspid aortic valve (n = 9), persistent left superior vena cava (n = 5), total anomalous pulmonary venous return or connection (n = 4), ‘heart valve problems’ (n = 3), coarctation of the aorta (n = 2), aortic stenosis (n = 2), enlarged aorta (n = 2), and enlarged aortic root (n = 1). Other congenital heart defects in the questionnaire (mitral valve prolapse) or cardiomyopathy were not reported in the study population.

Several dysmorphic features reported in PMS were associated with greater deletion sizes, including large fleshy hands (p = 9.47e−16, OR 1.50), sacral dimple (p = 5.05e−12, OR 1.45), dysplastic toenails (p = 1.56e−6, OR 1.24) or fingernails (p = 2.47e−3, OR 1.17), defective tooth enamel (p = 4.87e−3, OR 1.20), supernumerary teeth (p = 5.29e−3, OR 1.33), overgrowth (Too tall for age; p = 1.16e−3, OR 1.20), bushy eyebrows (p = 8.69e−3, OR 1.15), 2–3 toe syndactyly (p = 2.36e−2, OR 1.15), and diastema (Gap between two front teeth; p = 9.83e−3, OR 1.13). Recurring ingrown toenails were also more common in those with larger deletions (p = 8.65e−6, OR 1.29), probably related to toenail dysplasia. Teeth extractions were also more frequent (p = 8.94e−3, OR 1.18), possibly because of crowding and/or supernumerary teeth.

Abnormal brain magnetic resonance imaging (MRI) (p = 6.72e−5, OR 1.23) and computed tomography (CT) scans (p = 5.83e−4, OR 1.38), as well as the presence of specific abnormalities such as irregular brain ventricles, decreased gray matter, decreased myelination, and arachnoid cysts, were more likely to be found in patients with larger deletions (p values from 7.36e−4 to 2.40e−2, ORs from 1.20 to 1.32). Individuals with larger deletions also had more current seizures (p = 2.07e−2, OR 1.13) and more febrile seizures (p = 4.51e−6, OR 1.30). However, other types of seizures (e.g., absence seizures, tonic-clonic seizures, simple and complex partial seizures) were not associated with deletion size. Significant associations were observed for the need for intravenous antibiotics, chronic bronchitis, > 2 pneumonias in a year, > 2 serious sinus infections in a year, recurrent pneumonia, and placement of ear tubes (p values from 2.86e−4 to 3.98e−2, ORs from 1.16 to 1.31), suggesting that recurrent infections may be more frequent among individuals with larger deletions. Except for a mild association between deletion size and chronic aspiration (p = 1.90e−4, OR 1.26), no gastrointestinal conditions or symptoms were found to be associated with deletion size. The only ocular issue significantly associated with deletion size was strabismus (p = 1.16e−3, OR 1.19).

Phenotypes negatively associated with deletion size

Self-help skills had some of the strongest negative associations with deletion size, including Dresses without assistance, Undresses without assistance, Toilets independently, Night-time toilet trained, Eats independently, and Drinks independently (p values from 2.43e−9 to 2.75e−4, ORs from 0.327 to 0.558; p values and ORs for each phenotype are listed in Table 4). In addition, abilities related to socialization and imagination were more common among individuals with smaller deletions and SHANK3 variants: Awareness of imaginary characters, Engages in pretend play, Plays alongside others alone, Responds to others' emotions, Responds affectionately to caregivers, Hugs/kisses caregivers or dolls, Plays peek-a-boo, and Plays with peers (p values from 1.65e−5 to 3.09e−2, ORs from 0.327 to 0.750). Speech and language abilities (Number of words in a typical sentence, Verbal speech ability, and Ability to follow directions; p values from 9.41e−7 to 4.50e−3, ORs from 0.378 to 0.560) were observed more frequently in individuals with smaller deletions. Similarly, nonverbal communication skills, including Pointing and Gesturing, were used more often by individuals with smaller deletions (p values from 3.19e−3 to 4.87e−3, ORs from 0.855 to 0.860). Some speech disorders, such as Apraxia of speech, Receptive language disorder, and Expressive language disorder, were more frequently diagnosed in patients with smaller deletions (p values from 2.79e−4 to 6.31e−4, ORs from 0.759 to 0.807). Auditory processing disorder was also negatively associated with deletion size (p = 4.87e−3, OR 0.693), whereas no significant association was observed for hearing loss.

A range of psychiatric diagnoses, including autism, pervasive developmental disorder, attention deficit-hyperactivity disorder (ADHD), anxiety disorder, obsessive-compulsive disorder, and pica, were negatively associated with deletion size (p values from 4.51e−6 to 7.35e−3, ORs from 0.643 to 0.820). Attention deficit and hyperactivity symptoms were also less prevalent in individuals with larger deletions; specifically, Symptoms/diagnosis of ADD/ADHD, Acts as if driven by a motor, Runs around or climbs excessively when inappropriate, Appears to act without thinking, Difficulty playing quietly, Leaves seat when remaining seated is expected, and Difficulty sustaining attention in tasks (p values from 8.97e−8 to 4.06e−2, ORs from 0.486 to 0.721). Sleep disturbances (Difficulty falling asleep, Difficulty going back to sleep, Frequent nighttime awakenings, and Short nighttime sleep; p values from 9.40e−6 to 4.58e−2, ORs from 0.823 to 0.908) were all less frequent in individuals with larger deletions.

Interestingly, nearly all the cognitive development milestones surveyed showed significant impairment proportional to the size of the deletion. These included, Understands the use of familiar objects, Imitates household activities during play, Uses functional toys appropriately, Acts out recent experiences using gestures or words, Observes actions and imitates them later, and Understands the difference between food and objects (p values from 4.51e−6 to 4.87e−3, ORs from 0.786 to 0.864). Accordingly, regression in cognitive skills was observed less frequently in individuals with larger deletions (p value 1.99e−2, OR 0.862). Furthermore, Apgar scores at 1 and 5 min were significantly lower in individuals with larger deletions (p values 1.31e−2 and 1.35e−3, ORs 0.870 and 0.901, respectively), indicating an adverse impact on newborn health. Gestational age and birth weight were not associated with deletion size.

Finally, there were no associations with any endocrine (including hypothyroidism, too tall or too short for age, or puberty milestones), orthopedic (except scoliosis and torticollis), nose and throat, vision (except strabismus), or allergy-related conditions (Additional file 2: Table S1). Responses to sounds, movement, touch, oral input, or temperature variations were not associated with deletion size. Similarly, tolerance to pain was not associated with deletion size.

Genotype-phenotype associations in individuals with sequence variants or Class I or Class II deletions

Individuals with PMS can be classified as having SHANK3 sequence variants; small Class I deletions, including SHANK3 only or SHANK3 with ARSA and/or ACR and RABL2B; or larger Class II deletions, which include all other deletions [6]. The ARSA, ACR and RABL2B genes are not sensitive to haploinsufficiency and are therefore not expected to contribute to the phenotype of PMS. Comparison of individuals with sequence variants or Class I deletions versus those with Class II deletions, shown in Table 5, confirmed our findings based on the analysis of deletion size. Kidney abnormalities, lymphedema, congenital heart disease, abnormal brain imaging, hypotonia, feeding difficulties, and certain dysmorphic features were more common in individuals with Class II deletions than in those with sequence variants or Class I deletions. Individuals with sequence variants and Class I deletions were more likely to have verbal speech, self-help skills, and psychiatric diagnoses, including ASD, ADHD, depression, bipolar disorder and anxiety. Due to the nature of the parent-reported data used in this study, the frequencies in Table 5 are not directly comparable to those reported in studies based on clinical assessment or direct review of medical records [6]. In particular, the total number of individuals considered for each characteristic listed in the table includes the cases with a "No" response, but this does not necessarily mean that the clinical feature was analyzed in all individuals. There were also many unanswered questions, raising the possibility that some parents may have prioritized questions about their children's problems. For these reasons, we opted not to perform statistical analyses on these data.

Table 5 Clinical features of individuals with SHANK3 sequence variants, Class I deletions and Class II deletions