Additional file 1: Table S1. of Copy number variants and rasopathies: germline KRAS duplication in a patient with syndrome including pigmentation abnormalities

Gene content of the ~10.5-Mb chromosome 12p duplication between nt 23,728,536 and nt 34,189,943 (NCBI Build hg19), including 49 protein coding genes, two microRNA genes, and one long non coding RNA gene. (DOCX 24 kb

Splicing defect induced by the c.1048+5G>A mutation.

<p>(<b>A, B</b>) Schematic construction of pSpliceExpress reporter minigene containing exon 7, Wt and mutant, and their corresponding mRNAs. The position of the c.1048+5G>A mutation is indicated with a star (on the right). The first eight nucleotides downstream of the exon-intron junction are indicated. Abnormal longer transcript generated by the new donor splice-site mutation c.1048+5G>A is shown. (<b>C</b>) Electrophoresis of RT-PCR products obtained after transfection in HeLa cells of normal allele, c.1048+5G (lane 2) and mutant allele, c.1048+5G>A (lane 3) revealed on TapStation (Agilent). Samples were amplified using oligonucleotides complementary to exons 2 and 3 of insulin. The uppermost band in lane 2 of 525 bp corresponds to the normally spliced wt mRNA of the exon 7 with exons 2 and 3 of insulin. The uppermost band in lane 3 of about 560 bp correponds to the aberrant transcript resulted from a splicing failure and inclusion of an intronic part in the mRNA. The lower band in lane 2 and 3 corresponds to the band containing the exons of insulin alone without the exon 7 of ACVRL1. (<b>D</b>) Sequencing analysis of the longer transcript resulting from the mutant construct including 35 bp of the intron 7.</p

BMP9 response and western blot analysis of ALK1 known mutants.

<p>(A) Schematic localization of the 8 known mutations on the ALK1 protein. The exons in which the nucleic acid substitutions occur and their resulting amino acid substitutions are indicated. The mutations are distributed in the extracellular and the kinase domain of the ALK1 receptor. (B) Functional analysis of the ALK1 protein variants was performed by measuring luciferase reporter activity after BMP9 stimulation, as explained in materials and methods section. Results are expressed as fold induction over the value obtained for each ALK1 mutant in the absence of BMP9Data are mean ± SD of 3 independent experiments.(C) Western blot analysis of ALK1 protein variant was performed after transient transfection of HeLa cells by expression vectors encoding a C-terminally HA-tagged version of either Wt-ALK1 or the different novel ALK1 mutants. Transfection with an empty pcDNA3 vector was used as a control. After 48 hours, cells were lysed and 50 μg proteins of cell lysates were resolved by 10% SDS-PAGE and immunoblotted with antibodies against HA or against βactin as a loading control.</p

Normal splicing resulting from the c.1249A>T (Ile417Phe) mutation.

<p>(<b>A, B</b>) Schematic construction of pSpliceExpress reporter minigene containing exon 9, Wt and mutant, and their transcripted mRNAs. The position of the c.1249A>T (Ile417Phe) mutation is indicated with a star (on the right). The seven nucleotides around the mutation are indicated. A normal transcript generated by the mutant allele is shown. (<b>C</b>) Electrophoresis of RT-PCR products obtained after transfection in HeLa cells of normal allele, c.1249A (lane 2) and mutant allele, c.1249A>T (lane 3) revealed on 2% agarose gel. Samples were amplified using oligonucleotides complementary to exons 2 and 3 of insulin. The higher band in both cases of 380 bp corresponds to the normally spliced wt mRNA of the exon 9 with exons 2 and 3 of insulin (sequence not shown).</p

Functional and splicing defect analysis of 23 <i>ACVRL1</i> mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

<div><p>Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (<i>ENG</i>) and activin receptor-like kinase 1 (<i>ACVRL1</i>or <i>ALK1</i>) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in <i>ACVRL1</i> gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of <i>ACVRL1</i> induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted <i>ACVRL1</i> mutations.</p></div

Splicing defect induced by the c.733A>G (Ile245Val) mutation.

<p>(<b>A, B</b>) Schematic construction of pSpliceExpress reporter minigene containing exon 6, Wt and mutant, and their corresponding mRNAs. The position of the c.733A>G (Ile245Val) mutation is indicated with a star (on the right). The seven nucleotides around the mutation are indicated. The c.733A>G mutation induces the creation of a new donor splice site and the loss of the 3’ end of exon 6. (<b>C</b>) Electrophoresis of RT-PCR products obtained after transfection in HeLa cells of c.733A>G, mutant and normal alleles, revealed on 2% agarose gel. Samples were amplified using oligonucleotides complementary to exons 2 and 3 of insulin gene. The uppermost band of 397 bp in lane 3 corresponds to the normally spliced wt mRNA of the exon 6 with exons 2 and 3 of insulin. The band of about 357 bp in lane 2 correponds to the aberrant transcript resulting from a splicing failure and loss of an exonic part in the mRNA. The lower band in lane 2 and 3 corresponds to the fragment containing the exons of insulin alone without the exon 6 of ACVRL1. (<b>D</b>) Sequencing analysis of the short transcript that resulted from the mutant construct transfection, missing 40 bp of the exon 6.</p

Subcellular localization of ALK1 protein variants.

<p>HeLa cells were transiently transfected with the C-terminally HA-tagged ALK1 plasmids. Subsequently the permeabilized cells were co-stained with mouse monoclonal anti- HA antibody and with rabbit polyclonal anti- calnexin antibody and then processed for fluorescence confocal microscopy. In represented photos, the mutant Q111D reached the cell surface similarly to the ALK1-Wt. However the mutant L306P co-localized with the calnexin in the ER network was predominant (individual photos displaying each studied mutant ALK1 protein are presente in figure S1). Bars = 5μm.</p

BMP9 response and western blot analysis of ALK1 novel mutants.

<p>(A) Schematic localization of the 14 novel mutations on the ALK1 protein. The exons in which the nucleic acid substitutions occur and their resulting amino acid substitutions are indicated. The mutations are distributed in the extracellular and the kinase domain of the ALK1 receptor. (B) Functional analysis of the ALK1 protein variants was performed by measuring luciferase reporter activity after BMP9 stimulation, as explained in materials and methods section. Results are expressed as fold induction over the value obtained for each ALK1 mutant in the absence of BMP9. Data are mean ± SD of 3 independent experiments. (C) Western blot analysis of ALK1 protein variants was performed after transient transfection of HeLa cells by expression vectors encoding a C-terminally HA-tagged version of either WT-ALK1 or the different novel ALK1 mutants. Transfection with an empty pcDNA3 vector was used as control. After 48 hours, cells were lysed and 50 μg of cell lysates were resolved by 10% SDS-PAGE and immunoblotted with antibodies against HA or against βactin as a loading control.</p