Structure of the homeodomain.

<p>(A) Multiple sequence alignment among different species. Aminoacid residues involved in the deletion are included within a red box. (B) Three-dimensional representation of the interaction between the <i>Drosophila sp.</i> homeodomain and the target DNA sequence (orange). Aminoacids encompassed by the deletion are represented in green. C) Prediction of the electrostatic potential for the wild type protein (up) and the mutated one (down). Negative charge is represented in blue and positive charge in red.</p

<i>PHOX2B</i> deletion detected in exon 2.

<p>(A) Schematic representation of <i>PHOX2B</i> showing both nucleotide and amino acid sequence of exon 2; the 18bp corresponding to the deletion are boxed. (B) Sequence chromatogram of <i>PHOX2B</i> exon 2; the arrow indicates the beginning of c.393_410del18bp. (C) Pedigree and agarose gel electrophoresis of the amplification product of exon 2 in the patient and her parents.</p

Amplification and dHPLC conditions for <i>PHOX2B</i> genomic sequence analysis.

<p>Bp: size of the fragment; B: buffer concentration; CM: MgCl2 concentration; DNTPs: dNTPs concentration; P: primers concentration; Taq: units of Taq polymerase; DNA: DNA quantity; D: Denaturing temperature; DT: Denaturing time; H: Hybridization temperature; HT: Hybridization time; E: Extension temperature; ET: Extension time; T dHPLC: Injection temperature in dHPLC. The general PCR protocol was [95°C-5′]→[(D–DT) →(H–HT) → (E–ET)]×35cycles→[72°C–7′]→4°C.</p

Sequence variants detected in our cohort of HSCR patients.

<p>Compilation of all the sequence variants obtained through our genetic analyses.</p

Immunoblot analysis of NRG1 mutants.

<p>Human NRG1 wildtype (WT) and mutants (M111T, M139I and R438H) were overexpressed in COS7 cell line. A) The intracellular levels of NRG1 wild-type and M111T, M139I and R438H mutants were detected with anti-neuregulin 1α/ß1/2 (F-20) antibody. All three mutants showed a significant lower protein expression. Alpha-tubulin was used as the loading control for normalization. B) The <i>bar chart</i> represents the quantitative data of the relative protein expression levels normalized with alpha-tubulin in three independent assays. (**<i>p</i><0.005). C) The release of the extracellular domain in the medium was detected using anti-FLAG M2 antibody. The mutations did not affect the release of that domain of the NRG-1 protein. D) The <i>bar chart</i> represents the quantitative data of the relative protein expression levels normalized in three independent assays.</p

Analysis of cellular localization of NRG1 mutants.

<p>Human NRG1 wildtype (WT) and mutants (M111T, M139I and R438H) were overexpressed in COS7 cell line. Cellular localization of NRG1 wild-type and mutants were analyzed using immunostaining with anti-neuregulin 1α/ß1/2 (F-20) antibody. Immunosignal for the three mutants was different in comparison with WT. Patchier and perinuclear distribution was observed in the three mutants in comparison with the punctuate at cytoplasmic membrane distribution of the WT protein.</p

Candidate variants detected in HSCR patients.

<p>List of details of the candidate variants in <i>NRG1</i> isoform ß1 to be mutations associated with HSCR phenotype analyzed.</p>*<p>NA: Not available.</p><p>LS = Length segment.</p><p>Other = Mutations in other genes.</p

Allelic distribution and frequency of <i>NRG1</i> genotyped variants.

<p>Allelic distribution and frequency of the <i>NRG1</i> genotyped variants in HSCR patients and controls and their statistical comparison through <b>γ</b>².</p

Mutant NRG1 proteins.

<p>Scheme of the main regions of NRG1 isoform ß1 protein and the location of the three variants analyzed.</p

Liver stiffness progression rate according to <i>PNPLA3</i> rs738409 genotype.

<p>Liver stiffness progression rate according to <i>PNPLA3</i> rs738409 genotype.</p