Additional file 1: of Compound heterozygous variants in PGAP1 causing severe psychomotor retardation, brain atrophy, recurrent apneas and delayed myelination: a case report and literature review

Methods – Description of Quantitative magnetization transfer (MT) imaging, proton MR-spectroscopy, Whole exome analysis, RNA isolation and cDNA synthesis. (DOCX 19 kb

Sanger sequencing validation of the N309K variant.

<p>Sanger sequencing of the results for the proband, parent and unaffected control at nucleotide position 5: 95746646.</p

Homozygous variants within the diarrhea-linked regions of patient II-5.

<p>* Allele frequency (number of homozygous individuals) in the Exome Variant Server, NHLBI Exome Sequencing Project (ESP)²⁶.</p><p>REF – GRCh37 reference allele, ALT – variant allele, OMIM – Online Mendelian Inheritance in Man³⁶.</p><p>Homozygous variants within the diarrhea-linked regions of patient II-5.</p

Family Pedigree.

<p>Pedigree showing intractable diarrhea (green) and seizures (red) and other clinical phenotypes. A slash through the symbol indicates that the subject is deceased, and a double line between the parents indicates a consanguineous union.</p

Alignment of N309K missense variant to members of the PC1/3 gene family.

<p>The location of the missense mutation within a conserved region in the catalytic domain of the PC1/3 gene family.</p

Lack of activity of N309K PC1/3 despite robust secretion.

<p>PC/3-encoding vectors were transfected into HEK (A panels, <i>left side</i>) or Neuro2A cells (B panels, <i>right side</i>) and the conditioned media subjected to either enzymatic assay (A1, B1) using the standard fluorogenic assay; or to Western blotting using PC1/3 antiserum (A2, B2). Cell extracts were also subjected to Western blotting (A3, B3). Panel C shows the maturation of ³⁵S-methionine-labeled wild-type and N309K precursor proteins during a 20-minute pulse followed by a 2 h chase in Neuro2A cells. WT, wild-type PC1/3. Panel D shows a Western blot of Neuro2A cell lysate, previously transfected with either N309K and G593R PC1/3 cDNAs and incubated with α-FLAG M1 or α-FLAG M2 antibodies to discriminate the 94 kDa PC1/3 prodomain ER-retained form.</p

Homozygous regions linked to the enteric disease in the family.

<p>Homozygous regions linked to the enteric disease in the family.</p

LRRC6 expression in ciliated airway epithelial cells.

<p>Photomicrographs of normal human lung section (<b>A</b>) (scale bar = 100 µm) and bronchial epithelium (<b>B</b>) following immunofluorescent staining for LRRC6, which reveals the cytoplasmic localization of the protein (LRRC6, red) only in ciliated cells (acetylated α-tubulin, a cilia marker, green; DAPI: blue) (scale bar = 10 µm). Immunofluorescent staining of nasal epithelial cells cultured at an air-liquid interface from a healthy subject demonstrating cytoplasmic localization of LRRC6 (<b>C</b>) as compared to decreased expression in a cell from a PCD subject (<b>D</b>). LRRC6 was similarly present in a non-targeted shRNA (NT) treated airway epithelial cells (<b>E</b>) but absent in <i>LRRC6-specific shRNA</i> transfected cells (<b>F</b>) (scale bar = 10 µm). Ultrastructural appearance of cilia from a normal control (<b>G</b>), PCD subject (<b>H</b>)<b>,</b> and airway epithelial cells following transfection with non-targeted (<b>I</b>) and <i>LRRC6-</i>targeted (<b>J</b>) shRNA sequences. Arrow and arrow-head indicate inner and outer dynein arms, respectively.</p

Clinical characteristics of PCD subjects with <i>LRRC6</i> mutant alleles.

<p><b>Abbreviations:</b> BR, bronchiectasis; DC: dextrocardia; OM, chronic or recurrent otitis media; RS, rhinosinusitis; SI, <i>situs inversus totalis</i>; SS, <i>situs solitus</i>; ODA, outer dynein arm; IDA, inner dynein arm; ND, not done.</p

<i>LRRC6</i> relation to <i>Foxj1</i>.

<p>Immunoblot analysis of differentiating human tracheal epithelial cells collected from healthy subjects and grown at an air-liquid interface (<b>A</b>) showing LRRC6 paralleling Foxj1 expression. <i>LRRC6</i> expression measured using RT-PCR (<b>B</b>), increased significantly after the onset of ciliogenesis (ANOVA, p = 0.0004; individual student two-tail t-test, p = 0.003 and p = 0.001, comparing expression between day 0 and day 7, and between day 7 and day 14, respectively). <i>LRRC6</i> expression in tracheal airway epithelial cells isolated from wild-type mice (<i>Foxj1^+/+</i>) (<b>C</b>), compared to cells from Foxj1-deficient littermates (<i>Foxj1^−/−</i>) (<b>D</b>), showing that virtually no LRRC6 was detected in the cytoplasm of <i>Foxj1^−/−</i> cells. Immunofluorescent staining for LRRC6 (red) and α-tubulin (green). (scale bar = 10 µm).</p