Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy

Abstract

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with recurrent episodes of focal neuropathy primarily affecting the brachial plexus. Point mutations in the SEPT9 gene have been previously identified as the molecular basis of HNA in some pedigrees. However in many families, including those from North America demonstrating a genetic founder haplotype, no sequence mutations have been detected. We report an intragenic 38 Kb SEPT9 duplication that is linked to HNA in 12 North American families that share the common founder haplotype. Analysis of the breakpoints showed that the duplication is identical in all pedigrees, and molecular analysis revealed that the duplication includes the 645 bp exon in which previous HNA mutations were found. The SEPT9 transcript variants that span this duplication contain two in-frame repeats of this exon, and immunoblotting demonstrates larger molecular weight SEPT9 protein isoforms. This exon also encodes for a majority of the SEPT9 N-terminal proline rich region suggesting that this region plays a role in the pathogenesis of HNA.

Figure 1.

HNA-linked mutations are found in multiple SEPT9 transcripts. (A) The SEPT9 gene is approximately 220 Kb in size (hash marks denote 50 Kb intervals) and produces at least seven different mRNA transcripts encoding six different polypeptides. [Note, not all exons are to scale.] However, only SEPT9_v1, v2, v3, v5 and v6 contain known HNA mutations. Coding regions are indicated in black. HNA mutations are marked with asterisks. Two mutations are in both the coding regions of SEPT9_v1, v2, v3 (R88W and S93F) and the 5′-UTR regions of SEPT9_v5 and v6 (also known as v4* and v4). A third is located in the 5′-UTR of SEPT9_v3. (B) Domain structure of SEPT9 polypeptides. All SEPT9 isoforms posses a polybasic domain (shown in green) followed by a GTP-binding domain at the C-terminus. SEPT9 isoforms 1–3 also contain an N-terminal proline-rich extension. HNA mutations are indicated with arrowheads. Regions representing immunogens for SEPT9_i1-3, i5/6 and SEPT9_i1 antibodies are shown in black bars and labeled (1) and (2) respectively.

Figure 2.

Individuals possessing the shared haplotype express altered SEPT9 protein products. Lymphoblastoid cells from unaffected (K4035 and K4041), HNA affected founder haplotype (K4000, K4004, K4006, K4007, K4014, K4015, K4019, K4021, K4037, K4041 and K4042) and HNA affected point mutation (K4052 and K4018) individuals were lysed and probed with antibodies to SEPT9_i1-3 and i5/6 or SEPT9_i1 only. Individuals possessing the founder haplotype express novel SEPT9 reactive bands at ∼80 and ∼100 kDa. A SEPT9_i1 specific antibody shows that one of the bands at 100 kDa and one at 74 kDa are SEPT9_i1 reactive. Actin expression serves as a loading control.

Figure 3.

Altered SEPT9 protein products are a result of an in-frame tandem duplication of a 645 bp exon. (A) Primer sites in SEPT9 transcripts used for RT–PCR are indicated by arrowheads. (B) Lymphoblastoid cells from unaffected and HNA affected founder haplotype individuals express wild-type copies of SEPT9_v1, v2 and v3. (C) A minor band 650 bp larger than the expected wild-type product was often observed in founder haplotype individuals. This increase in size is the same as that of the exon containing previously identified HNA mutations. (D) PCR using primers within the 645 bp exon produces a larger minor band only in founder haplotype cDNA, suggesting two copies of the exon in tandem. (E) PCR using a reverse primer spanning the tandem duplication shows that individuals possessing the founder haplotype express SEPT9_ v1, v2 and v3 with two copies of the 645 bp exon. Forward primers in (E) are the same as those used in (A). (F) Model of SEPT9_i1 containing two copies of the 645 bp exon.

Figure 4.

Identification of a 38 Kb intragenic duplication within SEPT9. Array CGH results from two individuals possessing the HNA founder haplotype show a duplicated region within SEPT9. Dotted lines indicate proximal and distal breakpoints. The duplication encompasses the first exons of SEPT9 transcript variants 2 and 6 as well as the 645 bp exon in which two of the previous HNA mutations have been identified (marked with asterisks).

Figure 5.

Characterization of SEPT9 intragenic duplication. (A) To further refine the SEPT9 duplicated region, primers were designed to span the duplication junction. Sites of primers are noted in red and blue arrowheads. The presence of the tandem duplication yields a PCR product of ∼2 Kb. An exon outside of the duplicated region was used as a control (green arrowheads). The size of the SEPT9 gene containing the duplication is approximately 290 Kb. (B) Genomic DNA from unaffected (K4035 and K4041), HNA affected founder haplotype (K4000, K4004, K4006, K4007, K4014, K4015, K4019, K4021, K4037, K4041, K4042 and K4002) and HNA affected point mutation (K4052 and K4018) individuals was screened for the presence of the intragenic duplication. No LCLs were available for pedigree K4002. However, this family shares the common founder haplotype, and the same duplication was confirmed through genomic PCR. (C) Sequence analysis of the duplication breakpoint from two individuals with the founder haplotype. The highlighted nucleotide shows the proximal end of the duplication.

Figure 6.

Segregation analysis of founder haplotype family K4041. PCR analysis of the genomic breakpoint in all members of pedigree K4041 demonstrates consistent segregation of the disease with the duplication. Circles females, squares males, Empty symbols unaffected individuals, filled symbols affected individuals, cross-slashed symbols deceased, dotted symbols non-penetrant carriers.

Figure 7.

Affected HNA individuals in all shared haplotype pedigrees have an in-frame tandem duplication of the 645 bp exon. (A) To confirm the presence of in-frame tandem copies of the 645 bp exon, a forward primer (red arrowhead) at the 3′ end and a reverse primer (blue arrowhead) at the 5′ end of the exon were designed. The presence of tandem copies of the 645 bp exon yields a PCR product of 597 bp. Exons outside of the duplicated region were used as a control (green arrowheads). The wild-type size of the SEPT9_v1, v2 and v3 coding regions is 1685 bp plus alternate 5′ exons. The addition of the 645 bp exon in tandem increases this size to 2330 bp plus alternate 5′ exons. (B) cDNA from unaffected (K4035 and K4041), HNA affected founder haplotype (K4000, K4004, K4006, K4007, K4014, K4015, K4019, K4021, K4037, K4041 and K4042) and HNA affected point mutation (K4052 and K4018) individuals was screened from the presence of tandem copies of the 645 bp exon. (C) Sequence analysis confirms that the tandem duplication of the 645 bp exon remains in-frame. The highlighted nucleotide indicates the 3′ end of the exon.