Journal: Haematologica
Article Title: A mutation in the gene coding for the sialic acid transporter SLC35A1 is required for platelet life span but not proplatelet formation
doi: 10.3324/haematol.2018.198028
Figure Lengend Snippet: Blood and bone marrow smears, characterization of the SLC35A1 mutation in the family, and the capillary electrophoresis transferrin profile. Blood (A) and bone marrow (B) smears of patient II:1 were stained with May-Grünwald Giemsa reagent. The blood smear showed the presence of large platelets (relative to the size of red blood cells). The MKs from patient II:1’s bone marrow were immature (ii), as characterized by small size, hypolobulation and a basophilic cytoplasm, and (ii) granular MKs. C) The family’s pedigree, and Sanger sequencing results. Arrows indicate the position of the mutated nucleotide. Genetic mapping of disease loci in the family was carried out using an Affymetrix GeneChip Human Mapping 250K NspI SNP microarray. Multipoint linkage analysis of single-nucleotide polymorphism (SNP) data was performed using Alohomora and Merlin software. We performed homozygosity mapping of SNP data, and found 18 candidate loci of at least 1 Mb in size with a maximum logarithm of odds score (Zmax) of 1.8. Whole exome sequencing (WES) was performed in DNA from the index case (II:1), using the Exome Capture Agilent SureSelect XT V5 kit for library preparation and exome enrichment. Sequencing was performed on a Genome Analyzer IIx Illumina instrument in paired-end mode with a read length of 2x100bp. The median WES coverage was 60. Reads were aligned with the human reference genome sequence (UCSC hg19, NCBI build 37.3) using the BWA software package. Variants were selected using SAMtools, and then annotated using Annovar software. Variants in coding regions (including non-synonymous and nonsense mutations), intron-exon junctions or short coding insertions or deletions were selected when the minor allele frequency was less than 0.0030. The homozygous c.439T>C mutation in SLC35A1 was confirmed by Sanger sequencing, using primers flanking the mutations (SLC960-F: 5’-GCCCGGCCATTATCAAATA-3’, SLC960-R: 5’-AAATCAAAGAAATGTAGTCATGCTG-3’) in both affected individuals. Both affected individuals were homozygous for the mutation (II:1 and II:2, c.439T>C: p.Ser147Pro). The nucleotide and amino acid changes are indicated with respect to the reference sequences (Genbank: NM_006416.4 and NP_006407.1, respectively). Open symbols: unaffected; filled symbols: affected. D) The capillary electrophoresis transferrin profile for a control (top panel) and for patient II:1 (bottom panel) was performed as previously described.15 x axis: the migration time in arbitrary units. y axis: the optical density in arbitrary units. 5-sialo, 4-sialo, 3-sialo, 2-sialo, and 0-sialo: penta-, tetra-, tri-, di- and asialotransferrin, respectively. The isoform distributions for the control and for patient II:1 are indicated in the table.
Article Snippet: Genetic mapping of disease loci in the family was carried out using an Affymetrix GeneChip Human Mapping 250K NspI SNP microarray.
Techniques: Mutagenesis, Electrophoresis, Staining, Sequencing, Microarray, Software, Migration
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