Patients with 22q11.2 imbalance has broad prototypical variability, from no abnormalities to severe mental retardation with multiple congenital malformations. Because of the strong variance in phenotype, some patients with a deletion or duplication are not immediately identified or diagnosed at birth. In our study, MLPA P245 assay was applied to screen in the 6034 cases who presented with development retardation with or without other disorders. The results indicated that 22q11.2 is the third most frequent pathogenic CNVs with a frequency of 0.86%, following 15q11.2 (1.36%) and 7q11.23 (1.18%). The prevalence of 22q11.2 deletions and duplications were 0.61% (37/6034) and 0.25% (15/6034), respectively, which were similar to the data reported in the literature (0.69 and 0.36%) .
The high frequency of 22q11.2 copy number changes is attributed to the presence of a cluster of LCRs in 22q11.2 [11, 12]. There are eight LCR blocks named as LCR22 A to LCR22 H in the 22q11.2 region . Approximately 85–90% of individuals with 22q11.2 deletion syndrome have a 3 Mb deletion spanning from LCR22 A to D, while 8–10% have a nested 1.5 Mb deletion extending only from LCR22 A to B [13, 14]. In our study, among the 37 cases with 22q11.2 deletions, 26 (70.3%) cases carried LCR22 A-D deletions and 3 (10.8%) cases had LCR22 A-B deletions. We also identified 5 types of atypical deletions including LCR22 D-E (3/37, 8.1%), LCR22 C-D (2/37, 5.4%), LCR22 B-D (1/37, 2.7%), LCR22 E–F (1/37, 2.7%) and LCR22 B-F (1/37, 2.7%). The types of 22q11.2 deletions were more variable in our cohort and the prevalence varied largely, probably due to different sample size as well as our subjects selected as individuals with obviously developmental delay and/or mental retardation rather than congenital heart defects or craniofacial abnormality.
Studying the genotype and phenotype relationship of 22q11.2 deletion is difficult as the number of possible clinical symptoms is large and even individuals within families with the same type of deletion differ in clinical manifestation. Our subjects with LCR22 A-D deletion mostly presented with development delay, language delay, facial anomalies, heart defects, psychiatric/behavior problems, epilepsy and periventricular leukomalacia. Some of them also had hearing impairment, cryptorchidism etc. However, there was a 4 months old girl and a 7 years old boy who were requested by their parents for physical examination. Probably they had so mild symptoms that we couldn't identified, or they were too young to evaluate the associated phenotypes. They presented with no symptom at this stage. The underlying mechanisms of variable penetrance are not well understood at present. Many factors like genetic background, modifier genes, epigenetic changes and environmental factors may have important roles. More attention should be focused on the follow-up studies as the disorders like intellectual disability, psychiatric or behavioral problems may become obvious when they are older. Another case was a 27 years old woman, who didn't have clinical manifestation of her own. She could be classified as an asymptomatic carrier. Because of clinical variability and / or reduced penetrance, she was not identified until her three fetus were found to have congenital heart disease or hydronephrosis. As each child of an individual with 22q11.2 deletion syndrome has a 50% chance of inheriting the 22q11.2 imbalance. Therefore, genetic counseling for a pregnancy at increased risk, prenatal testing and preimplantation genetic testing are significant.
The symptoms of atypical deletions in our study mainly were language delay, development delay, intellectual disability, and growth delay. Because of the overlapping features of individuals with various 22q11.21 CNVs, the genotype–phenotype correlations could not be accurately predicted. Other factors that may impact the phenotypic similarity and variability remain to be determined. It has been reported that dysregulation of genes by loss of long-range regulatory sequences could affect either common genes and/or common developmental pathways [15, 16]. For example, long-range chromatin interaction of COMT in the proximal 22q11.2 region with genes on other chromosomes, as well as with genes in the distal 22q11.2 region may mediate similarities between typical, atypical, and distal 22q11 deletion phenotypes .
Of the 15 patients with 22q11.2 duplications, 8 had LCR22 A-D duplications, and the remaining 7 patients had LCR22 A-B (1/15, 6.7%), LCR22 C-D (1/15, 6.7%), LCR22 B-D (2/15, 13.3%), LCR22 E–F (1/15, 6.7%), LCR22 B-F (1/15, 6.7%) and LCR22 E–H (1/15, 6.7%), respectively. The types of duplications were also variable and the spectrum of symptoms was similar with 22q11.2 deletion syndrome. Our cases with LCR22 A-D duplications mainly presented with language delay, development delay, epilepsy, and periventricular leukomalacia. Intriguingly, we noticed that the 3 patients with only SNAP29 probe duplication in P245 assay, who were confirmed to have nested atypical duplications from C-D or B-D in P250 assay, presented with language delay, difficulty with social interactions and even autism. While the 3 patients with corresponding site deletion mainly showed development delay. A recent study concluded that the prevalence of neuropsychiatric disorders was higher in duplication carriers compared with deletion carriers . The atypical nested LCR22 B-D duplications are associated with an increased risk for neurodevelopmental phenotypes particularly autism spectrum disorder (ASD) ; suggesting the critical genes related with these neurodevelopmental phenotypes including ASD may be located between LCR22 B and LCR22 D. We performed NGS on the patient with LCR22 C-D duplication, who was clinically diagnosed with autism and found a 0.36 Mb duplication containing SNAP29 and SERPIND1 genes. SERPIND1 is very weakly expressed in brain tissues. Whereas SNAP29 has been found to negatively modulate neurotransmitter release and contributes to schizophrenia and autism spectrum disorder . It encodes a soluble NSF-attachment protein (SNAP) receptor (i.e., SNARE) protein that competes with a-SNAP for binding to SNARE complexes, thus reducing SNARE protein recycling and synaptic vesicle availability . Mutations in SNAP29 result in variable expressivity and incomplete penetrance. Patients with homozygous mutations in SNAP29 are responsible for CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma) syndrome , which has a number of clinical manifestations, some of which overlap with those found in 22q11.2 deletion syndrome. SNAP29 was reported to be abnormal in 90% of patients with 22q11.2 deletion syndrome. In our cohort, SNAP29 gene exhibited the highest frequency of variation among the 37 deletion and 15 duplication cases, with the incidence were 81.1% and 80%, respectively. Additional functional studies are necessary, to evaluate the role of SNAP29 in autism and the consequence of other genes expressed in LCR22 B-D in neurodevelopmental phenotypes.
In contrast to the prevalence of language delay, developmental retardation, cognitive impairment and behavioral problems in patients with LCR22 A-D and A-B imbalance, we found no incidence of cardiac defects and facial anomalies in any of our symptomatic patients with other atypical types of variation. We therefore postulated that the responsible genes for such factors are likely to lie within LCR22A to LCR22B region. Of note, HIRA, TBX1 and DGCR8 are considered candidate critical genes for major phenotypes associated with 22q11.2 disorders, especially the cardiac defects [22,23,24,25,26]. In addition, due to the fact that most of our patients were children, some clinical manifestations could be highlighted in the future such as phychiatric / behaviour problems, autism, headache, learning difficulties etc. So reassessment is required in these children in future follow up.