Identification and functional analysis of BICD2, a causal gene of autosomal dominant SMA

<p>Spinal muscular atrophies (SMAs) are characterized by degeneration of spinal motor neurons and muscle weakness. Autosomal recessive SMA is the most common form and is caused by homozygous deletions/mutations of the SMN1 gene. Additionally, dominant inheritance SMA families have been reported, for most of them the causal gene remains unknown. The starting point of the current study was a Dutch family (1) with non-progressive SMA, autosomal dominant (SMALED2; MIM #615290 AD) pattern of inheritance, unaltered SMN1, and congenital contractures. Linkage analysis and whole exome sequencing were performed and led to the identification of a heterozygous missense mutation (c.320C>T, p.Ser107Leu) in the BICD2 gene. BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D. BICD2 was sequenced in twenty additional families with SMALED2 finding additional mutations (p. Asn188Thr; p.Ala535Val; p.Thr703Met; p.Arg747Cys), and a rare variant p.90 Lys>Arg (VS frequency 0.4%) (5, 6). The patients differ in the severity of the symptoms, although some of those mutations lay on the same protein region. Overexpression of the mutant BICD2 cDNAs in HeLa cells was performed observing fragmentation of the Golgi Apparatus (GA). Fibroblast cell lines were derived from the patients carrying the mutations p.Thr703Met (severe phenotype) and p.Asn188Thr. A severe GA fragmentation was observed in the fibroblast cells from the patient p.Thr703Met suggesting a possible correlation between the grade of fragmentation and the severity of the disease. The integrity of the GA depends on the microtubule network and BICD2 is implicated in transport along microtubules. The microtubules of the fibroblasts carrying the p.Thr703Met were stained and alteration in their pattern was observed. BICD2 interacts with the small GTPase Rab6a (2); which plays an essential role in Golgi transport. Rab6a Pull Down analysis showed no alteration in the interaction of the BICD2 mutants and Rab6a. In order to understand the molecular mechanisms of the other mutations in BICD2, protein stability assays and additional interacting studies are being performed.</p> <p> </p

Workflow of the next generation sequencing strategies used.

<p>A) whole exome sequencing workflow. Samples have been pre-screened using an Apex-based Usher genotyping microarray; library preparations prior to enrichment include fragment single reads or Paired-End preparation. Three different types of enrichment methods have been used; each enrichment probe sets overlap at different extent to the RefSeq coding regions of Usher genes (horizontal bars). Sequencing protocols include single 50 bp reads on the Solid3 System, single 50 bp read on Solid4 System, Paired-end reads 50 bp+35 bp on Solid4 System. B) Long-PCR sequencing workflow. Samples have been pre-screened using Usher Apex microarray, Long-PCR approach produced 218 PCR amplicons used as input for the for <i>Fragment</i> and <i>Paired-End</i> library preparation. Sequencing was performed using both GS-FLX and GAII Systems.</p

Sequence variants of USH patients identified by whole exome sequencing.

(1)<p>We annotated the resulting variation according the USHbase database (<a href="https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html" target="_blank">https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html</a>) and the 9 variants not present in the database have been classified as unreported.</p>(2)<p>Clinical diagnosis compatible with USH type 3.</p

Coverage data for whole exome sequencing.

<p>A) Relationship between the minimum depth coverage and the extent of basepairs of RefSeq exons sequenced (shown in percentage). B) Relationship between the minimum depth coverage and the extent of basepairs of Usher exons sequenced. Solid colored lines represent different samples, x axis: minimum coverage increasing from left to right up to 50×; y axis: percentage of exons sequenced.</p

Coverage data for long-PCR NGS sequencing.

<p>Relationship between the minimum depth coverage and the extent of basepairs of Usher exons sequenced. Solid colored lines represent sample sequenced on different platforms, whereas the dotted line is the average representation obtained from the nine sample of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043799#pone-0043799-g001" target="_blank">Figure 1</a>. x axis indicates the minimum coverage increasing from left to right up to 50× while the y axis indicates the percentage of Usher exon basepairs sequenced.</p

Sequence variants of USH patients identified by Long-PCR sequencing.

1<p>Classification based on the USHbase database <a href="https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html" target="_blank">https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html</a>.</p

Segregation analysis of rare sequence variants identified in candidate genes in cuticular drusen (CD) families by whole exome sequencing (WES).

<p>Circles, females; squares, males; empty symbols, unaffected; black symbols, affected; asterisks, exome sequenced individuals; ‘+’ symbol, wild type allele; ‘m’ symbol, mutant type allele. The age at participation is specified below the symbols.</p

Recurrent missense variants identified in two of 289 candidate genes in 12 sporadic CD subtype of AMD cases by WES.

<p>Recurrent missense variants identified in two of 289 candidate genes in 12 sporadic CD subtype of AMD cases by WES.</p

Pedigrees of six cuticular drusen (CD) families in which whole exome sequencing (WES) was performed.

<p>Circle and square symbols indicate female and male individuals, respectively. Symbols with slashes indicate deceased individuals. Black and empty symbols indicate affected and unaffected individuals, respectively. Asterisks indicate the family members for whom exome sequencing was performed. The numbers below the symbols indicate the age at participation of family members.</p

Rare missense variants identified in known macular degeneration genes in 12 sporadic CD subtype of AMD cases by WES.

<p>Rare missense variants identified in known macular degeneration genes in 12 sporadic CD subtype of AMD cases by WES.</p