Diagnosing hereditary ataxias in a cohort of consanguine patients using a Next-Generation-Sequencing panel

<p>Background: Hereditary ataxias impose a relevant challenge when molecular diagnosis is sought. While more than 100 genes are involved in Mendelian diseases with ataxia, only a small proportion of these genes have been systematically tested in cohorts of patients with a consanguine family history. With the advent of next-generation-sequencing (NGS) a massive sequencing approach can be implemented with relatively ease. We investigated the occurrence of disease causing variants sequencing a cohort of closely related patients recruited for the EUROSCA and NEUROMICS EU projects respectively. The families originated mainly from the Mediterranean area. Each patient was strictly selected to avoid sequencing of persons suffering non hereditary kinds of ataxia or ataxia due to triplet repeat enrichment.</p> <p>Methods: We have established a selector-based enrichment method (HaloPlex, Agilent) specifically targeting 140 known ataxia genes as well as genes causal for rare diseases possessing a phenotypic overlap with ataxia. The panel covers most known genes causal for pure ataxia, mitochondrial ataxia and metabolic ataxia as well. A total of 582kb genomic DNA is specifically enriched and sequenced by Illumina MiSeq (2x 150 bp paired-end). Data analysis is accomplished using an in house bioinformatics pipeline based on ANNOVAR.</p> <p>Results: Although massive parallel sequencing usually brings up a couple of variants (Ø 384 ± SD 16), filtering for rare variants (in our own NGS database and in 1000g, ESP6500) and for functional relevance (ns,ss,indel) reduced this count to Ø 20 ± SD 4. A statistical evaluation of the panels performance shows superior coverage (Ø > 96 % cov 20X ± SD 1,8) and target enrichment values (Ø 178 ± SD 48 mapping depth on target) as well. Several disease causing mutations could be identified in genes like APTX, FGF14, NPC1, PLEKHG4, SACS, SETX, SIL1, SPTBN2, SYNE1 and many others.</p> <p>Conclusion: A panel sequencing approach offers a cheap and fast possibility to screen large patient cohorts for rare disease causing variants. Focusing on patients with a consanguine family background allows the discovery of rare and new variants for ataxia in a relatively high frequency.</p

MAN1B1-deficient individuals are deficient in protein N-glycosylation.

<p>MALDI-TOF spectra of the permethylated N-glycans from sera of control and MAN1B1-deficient individuals. Respective m/z values of N-glycans structures accumulating in MAN1B1-deficient cells are displayed in red. The symbols representing sugar residues are as follows: closed square, N-acetylglucosamine; closed circle: mannose; open circle, galactose; closed diamond, sialic acid; and closed triangle, fucose. Linkages between sugar residues have been removed for simplicity.</p

Overview of candidate variants identified by exome sequencing.

<p>% in the 1000 Genomes project were excluded. Based on recessive inheritance, a subsequent prioritization was applied using the following criteria: a minimum of 80% variant reads for potential homozygous and between 20 and 60% for compound heterozygous variants. For the analysis of candidate variants, variants from the single nucleotide polymorphism database (dbSNP) and with a higher frequency than 1</p

Clinical features of patients with MAN1B1 deficiency.

<p>+) or absence (−) of each feature is reported for each individual. The following abbreviations are used: NA, not available; yrs: years; F: female; M: male. The presence (</p

Functional analyses of <i>MAN1B1</i> mutations.

<p>(<b>A</b>) Quantification of the <i>MAN1B1</i> transcript in controls and affected individuals by qPCR without (left panel) and with (right panel) puromycin. <i>MAN1B1</i> expression was normalized to the expression of the house-keeping gene <i>HPRT</i>. Values plotted with a wild-type control untreated with puromycin are set to 1. The depicted values are the mean ± SEM of at least three independent experiments. (<b>B</b>) Steady-state levels of the expression of MAN1B1 in control (C) and MAN1B1-deficient (P) fibroblasts. Whole-cell extracts were analysed by immunoblotting using anti-MAN1B1 antibodies. Thirty micrograms of total cell extracts were loaded into each lane and anti-β-actin antibodies were used as a loading control. Quantifications represent the mean value of three independent loadings of three independent samples. (<b>C</b>) Intracellular distribution of MAN1B1 in control and MAN1B1-deficient fibroblasts. Fibroblasts were double-labelled with antibodies against MAN1B1 (green) and the Golgi marker giantin (red), then fixed and processed for analysis by confocal laser scanning microscopy. Images were collected under identical settings. Depicted are the zoomed images. The independent panels are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003989#pgen.1003989.s004" target="_blank">Figure S4</a>.</p

Clinical features of the seven cases.

<p>Clinical features of P1 at the age of 7 years (A), P2 at the age of 10 years (B, C), P3 at the age 3.5 years (D), P4.1 at the age of 18 years (E), P4.2 at the age of 12 years (F), P5 at the age of 13 years (G, H) and P6 at the age of 5 years (I, J). Note the facial dysmorphism, i.e. hypertelorism with downslanting palpebral fissures (A, D, E, F, G), large, low set ears (A, B, C, D, E, F, G, J), thin upper lip with hypoplastic nasolabial fold (A, B, D, E, F, G, I), tubular nose in P1, P4.1 and P4.2 (A, E, F) and a depressed nasal bridge in patients P2, P3, P5 and P6 (B, D, G, I). Note the truncular obesity (C, E, F, H) and the widely spaced, inverted nipples (B, F, H). Note the pectus excavatum in P5 (H).</p

Subcellular localisation of MAN1B1.

<p>(<b>A</b>) Indirect double immunofluorescence staining of control and MAN1B1-deficient fibroblasts. The cells were double-labelled with antibodies against MAN1B1 and with antibodies against either the Golgi markers GPP130 and giantin, the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53, or the ER marker PDI. The cells were then examined by confocal laser scanning microscopy. Images were collected under identical settings. Scale bar represents 10 µm. (<b>B</b>) Confocal linescan analysis of the distribution of MAN1B1. The pixel intensity (vertical axis) of MAN1B1 (green) and PDI, ERGIC-53, giantin and GPP130 (from top to bottom, red) was hence measured along a vector drawn perpendicular across the Golgi stack and plotted versus distance (horizontal axis), using the RGB profiler plugin from Image J.</p

MAN1B1-deficient fibroblasts present alterations in Golgi structure.

<p>Golgi localization of GM130 (red) and TGN46 (green) in control and MAN1B1-deficient fibroblasts. Cells were fixed, double-labelled with antibodies against GM130 and TGN46, and analysed by confocal laser scanning microscopy. Scale bar represents 10 µm.</p

IEF profiles of serum transferrin.

<p>Isoelectrofocusing pattern of serum transferrin in MAN1B1-deficient individuals and a control. 2, 4 and 6 indicate disialo-, tetrasialo-, and hexasialotransferrin isoforms, respectively. The asterisk mark represents the trisialotransferrin isoform.</p