FLNA genomic rearrangements cause periventricular nodular heterotopia

Abstract

Objective: To identify copy number variant (CNV) causes of periventricular nodular heterotopia (PNH) in patients for whom FLNA sequencing is negative.

Methods: Screening of 35 patients from 33 pedigrees on an Affymetrix 6.0 microarray led to the identification of one individual bearing a CNV that disrupted FLNA. FLNA-disrupting CNVs were also isolated in 2 other individuals by multiplex ligation probe amplification. These 3 cases were further characterized by high-resolution oligo array comparative genomic hybridization (CGH), and the precise junctional breakpoints of the rearrangements were identified by PCR amplification and sequencing.

Results: We report 3 cases of PNH caused by nonrecurrent genomic rearrangements that disrupt one copy of FLNA. The first individual carried a 113-kb deletion that removes all but the first exon of FLNA. A second patient harbored a complex rearrangement including a deletion of the 3' end of FLNA accompanied by a partial duplication event. A third patient bore a 39-kb deletion encompassing all of FLNA and the neighboring gene EMD. High-resolution oligo array CGH of the FLNA locus suggests distinct molecular mechanisms for each of these rearrangements, and implicates nearby low copy repeats in their pathogenesis.

Conclusions: These results demonstrate that FLNA is prone to pathogenic rearrangements, and highlight the importance of screening for CNVs in individuals with PNH lacking FLNA point mutations.

Figure 1. The FLNA locus is prone to rearrangement

(A) The FLNA and EMD genes are flanked by 11.3-kb inverted repeats at Xq28. FLNA encodes for the protein Filamin A, an actin cross-linking protein. EMD encodes for Emerin, a protein disrupted in Emery-Dreifuss muscular dystrophy. Recombination at this locus results in benign inversions. FLNA also lies telomeric to 3 low copy repeats of the color vision gene OPN1 and TEX28. Nonallelic homologous recombination–mediated deletion of OPN1-TEX28 repeats is a known cause of human color blindness. (B) Pedigrees of families 1, 2, and 3. Females are represented by circles, affected individuals by dark circles, and deceased individuals by an angled line. Arrows indicate the proband in each family. (C) Quantitative PCR (qPCR) confirmation of FLNA copy number loss in II-2 and I-2 of family 1. qPCR results targeting sequence centered on chrX:153,240,806. When normalized to a female control, it is apparent that the affected mother and daughter have one copy of the allele at that location, whereas the female control has 2 copies. The father, I-1, has one copy, which is expected for an unaffected male. The 95% confidence intervals were calculated using CopyCaller software by Applied Biosystems.

Figure 2. Brain MRI in affected individuals from families 1–3

Representative axial brain MRI images from affected individuals in this study. Bilateral gray matter nodules (arrowheads) line and project into the lateral ventricles, the classic appearance of periventricular nodular heterotopia. Family 1, I-2 and II-2, family 2, II-2: T1-weighted. Family 2, III-1, family 3, II-2 and III-1: T2-weighted.

Figure 3. High-resolution array comparative genomic hybridization (CGH) of the FLNA locus delineates distinct genomic rearrangements in each family with periventricular nodular heterotopia (PNH)

Top panel: A female individual with PNH, but without FLNA copy number change detected by quantitative PCR (qPCR), demonstrates signal intensities consistent with normal copy number across the locus. Second panel: Affected individual I-2 of family 1 demonstrates single-copy loss of all but the 5′ end of FLNA, as well as the adjacent TKTL1 gene. Third panel: Affected individual II-2 of family 2 demonstrates a single copy deletion of the 3′ end of FLNA, and duplication of the 5′ end of FLNA and EMD. Bottom panel: Affected individual II-2 of family 3 demonstrates deletion of both FLNA and EMD. The y-axis represents the log2 ratio of the signal intensity from the patient compared to a reference female control. Tall striped rectangles = inferred deletions; tall filled rectangle = inferred duplications; short striped and solid rectangles = regions of slightly decreased and increased signal intensity, respectively, across entire OPN1-TEX28 locus; green, red, blue shading = TKTL1, FLNA, EMD gene loci, respectively; gray hashes and short arrows = boundaries and direction of the inverted repeats flanking FLNA and EMD; gray vertical lines and long arrows = boundaries and direction of the OPN1-TEX28 tandem repeats. CNV = copy number variant.

Figure 4. Copy number changes at the FLNA locus in 3 families with periventricular nodular heterotopia implicate distinct molecular mechanisms

(A) Breakpoint sequencing supports a mechanism for family 1 rearrangement involving replication fork stalling and template switching/microhomology-mediated break-induced replication with template switches occurring between the second OPN1-TEX28 tandem repeat, the junction between the first and second OPN1-TEX28 tandem repeats, the third OPN1-TEX28 repeat, and FLNA intron 2. The result is loss of 1 OPN1-TEX28 tandem repeat and a deletion of all but the 5′ end of FLNA. (B) Breakpoint sequencing in family 2 supports 2 alternative models. In model 1, nonallelic homologous recombination between mispaired inverted repeats is followed by nonhomologous end joining (NHEJ) between 2 inverted copies of FLNA on opposite chromatids, leading to deletion of the 3′ end of FLNA and duplication of the 5′ end of FLNA and EMD. Model 2 presupposes a heterozygous background for the benign FLNA-EMD inversion, in which case a single NHEJ event between FLNA intron 41 on one chromatid and FLNA exon 20 on the second chromatid is sufficient to generate the observed rearrangement. (C) A speculative, replication-based model for FLNA rearrangement in family 3, in which the inverted repeats flanking FLNA and EMD form a single-strand secondary structure that results in contiguous deletion of both genes and the inner half of both repeats.