We report a 2p13.1-p13.3 microdeletion observed in two subjects with clinical effects on the cognitive function (ID and language delay), behaviour (hyperactivity), and development of the craniofacies (facial asymmetry, unusually shaped and asymmetric ears and brachycephaly). Skull and vertebral bone abnormalities included slight asymmetry of the appearance of the orbits and delayed closure of the metopic suture in Subject 1 and congenital C1-C2 vertebral fusion, and accentuation of dorsal kyphosis in Subject 2. Previous cases with cytogenetic deletions/disruptions of this region all had developmental delay and the majority had head/facial anomalies, ear and skeletal abnormalities [1, 2].
The overlapping deleted region of our two cases encompasses two genes, EXOC6B and CYP26B1, both of which showed altered function in whole blood and/or LCLs from Subject 1. The reduced expression of EXOC6B in LCLs and whole blood could be the cause of the observed perturbed Notch signaling (i.e. HES1 and RBPJ expression change), considering the similar effect of EXOC6B knockout on Notch signaling in Drosophila.
20 The reduction of RNA expression of EXOC6B and RBPJ was concordant between LCLs and whole blood, and the expression of the RBPJ protein also was reduced in Subject 1 lymphoblasts. However, HES1 had reduced RNA and protein expression in LCLs and increased RNA expression in whole blood. The discrepancy in the pattern of abnormal expression of HES1 in different cell types could be due to the difference in the Notch signaling pathway in lymphoblasts which contain dedifferentiated B cells vs. whole blood which contains multiple differentiated cell lineages. Cell-specific over or underexpression of Hes1 has been reported in different regions of the brain in Rbpj knockout mice .
EXOC6B germline mutations or deletions have yet not been reported in humans. A homozygous mutation in EXOC6B’s paralogue EXOC6A has been reported in mice with hemoglobin-deficit (hbd) due to defective iron transport in the endocytosis cycle  while haploinsufficiency of EXOC6A due to a 0.3 Mb microdeletion at 10q23.33, was reported in a family with nonsyndromic bi- and unilateral optic nerve aplasia . Interestingly, this microdeletion also included two CYP genes, CYP26A1 and CYP26C1. In contrast to very little information on genetic defects of EXOC6B and their role in disease, genetic abnormalities of HES1 and RBPJ have been associated with a number of developmental defects in vertebrates. Homozygous Hes1 and Rbpj knockout mice showed severe developmental defects and lethality [43, 44], while Rbpj heterozygous knockout mice demonstrated learning deficits . In humans, increased expression of HES1 was reported in Down syndrome  and recent exome sequencing studies revealed heterozygous mutations in RBPJ and reduced expression of HES1 in two families with Adams-Oliver syndrome, associated with congenital cutis aplasia, terminal limb abnormalities (asymmetric shortening of the hands and feet in one of the families) and a range of cognitive functioning (from intellectual disability to normal) .
The deletion of CYP26B1 gene in both our Subjects is also likely to contribute to their abnormal phenotype, based on abnormal RA metabolism in Subject 1, as evidenced by significantly attenuated induction of CYP26B1 expression with ATRA in comparison to controls. To the best of our knowledge, there are no reports on the effect of CYP26B1 gene haploinsufficiency in humans. Previously, in two other families, two different homozygous mutations of CYP26B1 have been reported, resulting in lethality, skeletal and craniofacial abnormalities, including fusion of long bones, calvarial bone hypopasia and craniosynostosis . In one of the families with the hypomorphic mutation, brachycephaly and wide sagittal sutures were noted. The two mutation-bearing constructs had attenuated ability to metabolize ATRA (36% and 86%) . In our Subjects, the decreased RA catabolism and increased sensitivity to RA, as a consequence of CYP26B1 deletion could explain features similar to those noted by Laue et al.  (e.g. brachycephaly for both subjects, and for Subject 1, the delayed closure of the metopic suture) and in general compromise the craniofacial, skeletal development and neuronal functioning. With regard to the later phenotype, it is of interest that Subject 1 had an asymmetric crying face as a consequence of a right facial nerve palsy which previously was associated with RA exposure or early embryonic insult [11, 47]. The phenotype of the subjects is in agreement with developmental effects of CYP26B1 deletion in experimental animals . CYP26B1 null mice have craniofacial abnormalities, exhibit abnormal ear development and other bone and cartilage deformities . Interestingly, the truncated limbs observed in CYP26B1-/- mice were absent in the patients. Similarly, in the zebrafish CYP26B1 deletion has been shown to result in overall defective craniofacial cartilage development with smaller head, severely decreased number of vagal branchiomotor neurons and defective or absent jaw cartilage .
It is intriguing to note the lethal phenotype in two subjects reported by Laue et al. due to homozygous mutations of CYP26B1 in comparison to the survival and developmental abnormalities in our subjects with hemizygous CYP26B1 deletion. The presence of one normal copy of the gene in each of our subjects and the efficiencies of the other two remaining RA catabolizing CYP26 genes (CYP26A1 and CYP26C1), possible compensatory changes in other proteins known to regulate retinoic acid, such as RALDH [8, 49, 50] and environmental influences, such as diet and pharmacological treatment, [6, 51] all could have an effect on the phenotypes. Phenotypic variability also was noted for carriers of CYP26A1, CYP26C1 and EXOC6A deletions within one family , ranging from normal vision, to uni- and bilateral optic nerve hypoplasia and variable levels of cognitive functioning (normal to impaired).
The combined effect of deletion of both EXOC6B and CYP26B1 on Notch and RA signaling, and consequently the phenotype, also should be considered in our subjects. Interaction of RA and Notch signaling in determination of left/right asymmetry and segmentation has been reported by Echeverri and Oates  who noted the requirement of Rbpj function for expression of RA catabolizing enzyme Cyp26a1 which in turn, is needed for left/right symmetric cyclic gene expression. Vermot et al. demonstrated that reduced levels of RA were associated with abnormal Hes1 expression and asymmetry in mouse embryos, while Castella et al.  showed that addition of RA raises the level of HES1 protein expression in in vitro cell culture.
Subject 2 had additional phenotypic features not noted in Subject 1, which might be explained by the larger size of the 2p13.1-13.3 deletion, interaction of its integral genes and genetic background. For example, seizure-like episodes were not noted in Subject 1, but were present in Subject 2 and a Decipher subject, # 257412, with developmental delay, whose deletion of 2p12-13.3 was 6.8 Mb (70,889,254-77,746,500), and his features included coarse faces, a flat malar region, myopathic hypotonia and prominent ears. The cervical fusion and spine abnormality are unique for Subject 2 and are of interest, considering the report of a subject with Klippel-Fiel anomaly, who had a balanced inversion involving chromosome 2p13 and congenital fusion of the cervical spine, impairment of hearing, psychomotor retardation, speech limitation, short stature, spinal asymmetry and scoliosis . The genes disrupted/deleted by this chromosome rearrangement are unknown, however, it was speculated that CYP26B1 was involved based on the similar vertebral phenotype observed in zebrafish with Cyp26b1 mutations .
Our report is unique as it provides a new insight into the phenotypic and functional consequences of hemizygous deletion of two genes implicated in Notch and Retinoic acid signaling. It also supports the previous cell line and animal model based observation of exocyst role in Notch signaling. Further studies of exocyst complex function in patient cells would be of interest for understanding of its role in human disease.