In the literature, there are eight previously reported cases carrying 3q26-3q28 microdeletions with sizes of 2-8.4 Mb that overlapped with the deleted chromosomal regions in two patients from this study [7–12]. The clinical phenotype of individuals with 3q26-3q28 microdeletions is heterogeneous: IUGR, severe growth impairment, feeding problems, short stature, dysmorphic facial features, microcephaly, seizure, eye and ear abnormalities, clinodactyly, feet abnormalities, developmental delay, intellectual disability, hypotonia, and thrombocytopenia. While there is some degree of phenotypic variability that primarily relates to the size of the deletion, the most striking clinical features shared among all reported cases are severe prenatal and postnatal growth restriction, as well as neurodevelopmental abnormalities. The clinical presentation of two patients described in this study supports the clinical profile described for other individuals in the literature (Table 1). The genotype-phenotype correlations for loss of the 3q26q28 region are, however, restricted by the fact that these individuals do not share common break points, like those generated in recurring pathogenic CNVs flanked by segmental duplications. Nonetheless, comparison of the clinical and molecular findings in Proband-1 and Proband-2 with the previous reported individuals suggests that this is a clinically recognizable microdeletion syndrome with shared clinical features. Though the precise size and position of these deletions are uncertain, it has been proposed that haploinsufficiency of dosage sensitive genes leads to defined clinical sequelae [7].
By comparing microarray findings of these ten cases, we mapped the SRO to a size of 1.2 Mb, corresponding to genomic coordinates 183,220,510 − 184,469,308 (hg19) at chromosomal band 3q27.1. This SRO region contains 46 genes, 24 of which are OMIM-annotated and eight of which are associated with disease (KLHL24, EIF2B5, DVL3, AP2M1, ALG3, EIF4G1, CLCN2, and THPO) (Table 2). Among these genes, DVL3 is the most interesting one. Heterozygous pathogenic variants in the DVL3 gene have been associated with autosomal dominant type III Robinow syndrome (MIM#: 616894), which shares many clinical features with the 3q26q28 microdeletion syndrome: short stature (9/9), facial dysmorphic features (10/10), epicanthal folds (7/9), nasal features (9/10), teeth abnormalities (7/8), hand abnormalities (4/10), clinodactyly (6/10), genital/urinary abnormalities (5/10). Emerging data suggest DVL3 is a core component in the routing and transmission of canonical and non-canonical Wnt signalasome [14]. In murine, DVL3 has been detected to express ubiquitously at E7.5, but shortly after it showed elevated expression in heart, CNS, notochord, dorsal root ganglia, branchial arches, limb buds, and somitic mesoderm [15, 16]. These findings further strengthen the role of DVL3 in diseases by modulating Wnt signaling that is involve in cell migration and tissue morphogenesis in vertebrate development. Indeed, Dvl3 knock out mice demonstrated partial lethality, conotruncal defects and neural tube defects, including abnormalities in cochlear cells [17]. However, all current known pathogenic variants of DVL3 are frameshift small insertions/deletions or splice variants in the last two exons; and larger intergenic deletions of DVL3 have not been described previously [18–20]. Furthermore, it was demonstrated by expression studies that truncating DVL3 variants escape nonsense-mediated decay (NMD), suggesting a dominant-negative or gain-of-function disease mechanism [19–22]. Therefore, the exact contribution from loss of DVL3 to phenotype caused by 3q26-3q28 microdeletions is still uncertain at this time.
Beside DVL3, the role of AP2M1 (MIM#: 601024) in poor speech (7/9), developmental delay (9/9), hypotonia (6/9), and seizures (2/9), as well as the role of PARL (MIM#: 607858) in growth restriction (10/10) are of great interest. AP2M1 has recently been associated with impaired intellectual development, poor speech and delayed walking [23]. Though a recurrent missense variant in AP2M1 has been reported, AP2M1 is highly intolerant to loss-of-function variant in general population with a probability of intolerance to loss of function (pLI) of 1.0 and the Haploinsufficiency Score of 8.13. Previous studies with Parl knock out mouse model have shown that Parl plays an essential physiological role in the neurological homeostasis [24], and Parl deficiency results in growth retardation, cachexia, and severe atrophy of skeletal muscle, thymus, and spleen [25]. However, we think the growth phenotype caused by this SRO is predominantly overlap with DVL3 related Robinow syndrome and further study is warranted to associate the role of PARL in this phenotype. The remaining OMIM genes in the SRO (ALG3, CLCN2, EIF2B5, EIF4G1, KLHL24, and THPO) are associated with autosomal recessive conditions and therefore are less likely to have major contributions to these patients’ phenotype.
In order to evaluate the clinical significance of the SRO, we further assessed the deduced SRO corresponding to genomic coordinates, chr3:183,220,510 − 184,469,308 (hg19) using ACMG/ ClinGen current recommendations for classifying copy number variants (CNVs) [13]. This SRO harbor 26 protein-coding RefSeq genes (Criteria 3B, points given: 0.45). Three of them (PSMD2: pLI:1; HI%: 6.89; AP2M1: pLI:1; HI%: 8.13; EIF4G1: pLI:1; HI%: 9.34) have predicted haploinsufficiency score below 10% and loss intolerance (pLI) score of 1 (Criteria 2H, points given: 0.15). To the best of our knowledge, in the literatures the SRO overlap with four previously assumed (due lack of molecular confirmation for paternity and maternity) de novo cases (individuals 1, 2, 5, and 7 in Table 1) with phenotype that is consistent with the gene/genomic region, but not highly specific and/or with high genetic heterogeneity (Criteria 4C, points given: 0.40). Moreover, observed copy number loss is assumed de novo (due lack of molecular confirmation for paternity and maternity) for Proband-1 in this study (Criteria 5A, points given: 0.1). Using these recommendations as a framework, we classified the SRO as pathogenic (Total score: 1.1).
In conclusion, in this study we present two additional individuals with phenotype similar to previously reported cases with overlapped deletions. It provides further evidence supporting the existence of this novel 3q26q28 microdeletion syndrome. Additionally, our molecular cytogenetic and clinical findings defined the 1.2 Mb SRO at chromosomal band 3q27.1 as the critical region for this clinically recognizable syndrome. The refinement of this critical region suggests that deletion of at least three genes (DVL3, PARL and AP2M1) may contribute to anomalies observed in these individuals. At last, we demonstrated that application of new ACMG/Clingen standards for CNV interpretation with refined molecular mapping would improve our ability for clinical diagnosis and genetic counselling of individuals harboring similar imbalance.