Risk of meningomyelocele mediated by the common 22q11.2 deletion

Abstract

Meningomyelocele is one of the most severe forms of neural tube defects (NTDs) and the most frequent structural birth defect of the central nervous system. We assembled the Spina Bifida Sequencing Consortium to identify causes. Exome and genome sequencing of 715 parent-offspring trios identified six patients with chromosomal 22q11.2 deletions, suggesting a 23-fold increased risk compared with the general population. Furthermore, analysis of a separate 22q11.2 deletion cohort suggested a 12- to 15-fold increased NTD risk of meningomyelocele. The loss of Crkl, one of several neural tube-expressed genes within the minimal deletion interval, was sufficient to replicate NTDs in mice, where both penetrance and expressivity were exacerbated by maternal folate deficiency. Thus, the common 22q11.2 deletion confers substantial meningomyelocele risk, which is partially alleviated by folate supplementation.

Fig. 1.. Deletions in chromosome 22q11.2 associated with MM.

(A) Trios of MM were recruited to the SBSC from multiple countries, including the United States, Mexico, Brazil, Canada, Italy, Georgia, Egypt, Nigeria, and Pakistan. Red dots indicate city locations of recruitment centers. DNA samples from trios were evaluated by whole-exome sequencing and/or whole-genome sequencing to identify de novo or inherited genetic variants. SNP, single-nucleotide polymorphism. (B) The standard and critical deletions of 22q11.2 LCRs (LCR22) for DiGeorge risk span 2.5 or 1.5 Mb, respectively (blue bars). Six patients with MM (P1 to P6, cohort size n = 715) showed chromosomal 22q11.2 deletion (22q11.2del, light blue bars): four de novo (P1, P2, P4, and P5) and two inherited from a healthy parent (P3 and P6). Whereas approximately 85% of 22q11.2del spanned the classic LCR22A-D region (35), two-thirds of MM cases showed partial deletions, either LCR22B-D or LCR22C-D (P3 to P6). Another three MM patients with 22q11.2del were referred to our study (purple bars, AP1 to AP3): two siblings with an inherited LCR22B-D deletion [AP1 and AP2, as previously described (14)] and a patient with an LCR22C-D deletion (AP3). The consensus LCR22C-D deletion interval in all MM patients includes 10 protein-coding genes. In addition, nine MM patients (yellow bars) were identified, eight from the Children’s Hospital of Philadelphia (CHOP) and one from Spain (madrid_660), which is part of the International 22q11.2 Brain and Behavior Consortium, all of whom had LCR22A-D deletions. Only patients from CHOP were included for odds ratio calculation.

Fig. 2.. NTDs in Crkl mutants are exacerbated by a FA-restricted diet.

(A) Brightfield images showing gross morphology of control and Crkl null mutants that displayed a curly tail. White arrows identify the caudal neural tube and tail in control and Crkl^−/− embryos. (B) Brightfield images of Crkl mutant embryos with curly tail (white arrows) and open neural tissue (white arrowheads) showing MM. (C) Brightfield image of Crkl mutant with exencephaly, a severe form of NTD. The white arrow identifies exposed brain tissue. (D) Penetrance of NTD phenotypes across genotypes and FA-diet treatments. No controls (Crkl^+/+ and Crkl^+/−) displayed NTD phenotypes on either a normal or a low-FA diet. With control diet (FA at 3 ppm), Crkl^−/− mutants displayed curly tail at 7.46% penetrance (5/67) or, rarely, curly tail together with MM (2.99%, 2/67) or exencephaly (1.49%, 1/67). The penetrance for exencephaly increased significantly in Crkl^−/− mutants after a FA-restricted diet (37.5%, 3/8, odds ratio of 34.96, P = 0.0031, 95% CI = 3.05 to 400.71), whereas curly tail or MM was not observed (see also fig. S8).

Fig. 3.. Signaling defects in Crkl mutant neural tube.

(A) CRKL immunostaining in E9.5 mouse showing broad expression in neural tube. Insets i and ii show corresponding magnified areas. (B) Phosphorylated ERK1/2 immunostaining of E9.5 mouse neural tube of control and Crkl^−/− embryos. Insets show high magnification of the ventricular zone. The bar graph shows the quantification of ERK1/2 phosphorylation cells within the ventricular zone, from at least eight images of two animals per genotype (P = 0.0062). Error bars indicate standard deviation. (C) c-Abl localization in control predominantly excluded from the nucleus (white arrows in inset i) versus greater accumulation in the nucleus (yellow arrows in inset ii) in Crkl^−/−. Insets i and ii show corresponding magnified areas. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars are 20 μm in (A) and 50 μm in (B) and (C).