Genomic analysis identifies masqueraders of full-term cerebral palsy

Abstract

Objective: Cerebral palsy is a common, heterogeneous neurodevelopmental disorder that causes movement and postural disabilities. Recent studies have suggested genetic diseases can be misdiagnosed as cerebral palsy. We hypothesized that two simple criteria, that is, full-term births and nonspecific brain MRI findings, are keys to extracting masqueraders among cerebral palsy cases due to the following: (1) preterm infants are susceptible to multiple environmental factors and therefore demonstrate an increased risk of cerebral palsy and (2) brain MRI assessment is essential for excluding environmental causes and other particular disorders.

Methods: A total of 107 patients-all full-term births-without specific findings on brain MRI were identified among 897 patients diagnosed with cerebral palsy who were followed at our center. DNA samples were available for 17 of the 107 cases for trio whole-exome sequencing and array comparative genomic hybridization. We prioritized variants in genes known to be relevant in neurodevelopmental diseases and evaluated their pathogenicity according to the American College of Medical Genetics guidelines.

Results: Pathogenic/likely pathogenic candidate variants were identified in 9 of 17 cases (52.9%) within eight genes: CTNNB1,CYP2U1,SPAST,GNAO1,CACNA1A,AMPD2,STXBP1, and SCN2A. Five identified variants had previously been reported. No pathogenic copy number variations were identified. The AMPD2 missense variant and the splice-site variants in CTNNB1 and AMPD2 were validated by in vitro functional experiments.

Interpretation: The high rate of detecting causative genetic variants (52.9%) suggests that patients diagnosed with cerebral palsy in full-term births without specific MRI findings may include genetic diseases masquerading as cerebral palsy.

Figure 1

Patient flowchart. CP, cerebral palsy; CPA, cardiac pulmonary arrest.

Figure 2

Schematic map of variants filtering. Rare variants and candidate variants in this figure are shown in Table S1 and Table 2, respectively. GQ, genotype quality; AAR, alternative allele ratio; AR, autosomal recessive; XL, X‐linked; ACMG, the American College of Medical Genetics and Genomics* “Databases” include the 1000 Genomes database (all and East Asian populations), the ESP6500, the ExAC database version 0.3 (all and East Asian populations) and the HGVD.

Figure 3

Nonspecific brain findings on T2‐weighted MRI. (A) Case 5; mild hyperintensity of the deep white matter around the body of the left lateral ventricle. (B) Case 7; slight hyperintensity of the left pars triangularis. (C) Case 11; hyperintensity in the striatum and a thin corpus callosum. (D) Case 13; a thin corpus callosum and mildly enlarged lateral ventricle. (E) Case 14; a thin corpus callosum and cerebral white matter atrophy. (F) Case 16; slight bilateral hyperintensities in the pallidum.

Figure 4

Analysis of the splice‐site variant of CTNNB1 in case 3 and AMPD2 in case 11. (A) RT‐PCR product of partial CTNNB1 run on 2% agarose gel electrophoresis. RT‐PCR products obtained from RNA of peripheral blood lymphocytes. (B) CTNNB1 sequence analysis of cDNA. The locations and corresponding cDNA sequencing results focusing on the exon 10–11 junction in the wild‐type (top) and CTNNB1 mutant cDNA (bottom) obtained from RT‐PCR. The corresponding translated amino acid sequences for the actual and hypothetical mutant products are also shown. The locations of the variants and stop codons are marked in the mutant diagram. (C) RT‐PCR product of partial AMPD2 from electrophoresis. (D) AMPD2 sequence analysis of cDNA. The locations and corresponding cDNA sequencing results focusing on the exon 4–5 junction in the wild‐type (top) and AMPD2 mutant cDNA (bottom) obtained from RT‐PCR. The corresponding translated amino acid sequences for the actual and hypothetical mutant products are also shown. The locations of the variant and stop codons are marked in the mutant diagram. bp, base pair; MW, molecular weight; NC, negative control; Ctr, control.

Figure 5

AMP deaminase assay. (A) Blue intensity represents relative activity. NC, Negative control; Ctr, Control. (B) Quantification of relative absorbance ± SD for six independent experiments measured at 625 nm. *P < 0.001. a Dunnett's test for unequal variance.