Family structures for 23 leprosy families.

<p>Family structures for 23 leprosy families.</p

Genome-wide linkage results.

<p>Genomic position is shown on the horizontal axis; HLOD(parametric Model)/LOD(non-parametric model) score on the left vertical axis.; Information content on the right vertical axis. Red line indicates results under a recessive model; blue line indicates a dominant model, dashed line indicates nonparametric analysis, and green line indicates information content.</p

Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify <i>ABCB6</i> as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria

<div><p>Background</p><p>As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH.</p><p>Methodology</p><p>We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation.</p><p>Results</p><p>Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified <i>ABCB6</i> as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of <i>ABCB6</i> in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of <i>ABCB6</i> in melanocytes and pigmentation. Given the involvement of <i>ABCB6</i> mutations in coloboma, we performed ophthalmological examination of the DUH carriers of <i>ABCB6</i> mutations and found ocular abnormalities in them.</p><p>Conclusion</p><p>Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma.</p></div

Number of mature melanocytes in the head region in the morphants and rescued embryos (5 dpf).

<p>Immobilized embryos were dorsally oriented and the number of mature melanocytes was counted in the defined region of the head. Reduction in melanocyte number was observed in both exon 6- and exon 8-morphants (B and E). Co-injection of wildtype <i>hABCB6</i> mRNA transcript significantly rescued the loss of melanocyte phenotype by increasing the number of mature melanocytes (C and F).</p

Phenotypes of the microinjected zebrafish.

<p>Morpholinos against exon 6 and exon 8 of <i>zABCB6</i> and wildtype <i>zABCB6</i> mRNA transcript (in case of rescue) were designed and injected into one- to two-cell stage embryos. No obvious phenotypic changes were observed in the morphants (C and D) and rescued (E and F) embryos after 5 days (5pdf) with reference to their uninjected counterparts (A and B).</p

Family trees and clinical manifestations.

<p><b>A</b> Shown are the pedigree of family 1 and 2 with autosomal dominant DUH. “#”represents the individuals used in exome sequencing analysis, “☆”represents the individuals used in the linkage analysis; “▴”represents the individuals subjected to Sanger sequencing analysis. <b>B</b>: Clinical manifestations of DUH patients, both hands and abdomen with hyper- and hypo-pigmented macules in variable distribution and patterns.</p

Number of melanocytes in the defined head regions in uninjected control (wildtype), exon 6- and exon 8-morphants and rescued embryos.

<p>**P<0.001 (ANOVA one-way analysis of variance with Tukey’s multiple comparison post test).</p

Mutations in ABCB6.

<p><b>A:</b> Two mutations in <i>ABCB6</i> and their sequencing traces, including c.1358C>T; p.Ala453Val in the Family 1 and c.964A>C; p.Ser322Lys in the Family 2; Arrows indicate the location of the two mutations. <b>B:</b> A partial sequence of ABCB6 was compared with other species’ orthologs. Arrows indicate the location of the two mutations identified in patients with DUH. <b>C:</b> <i>ABCB6</i> exon structure, where c.964A>C; p.S322L and c.1358C>T; p.A453V are DUH mutations identified in this paper, c.169G>A; p.A57T and c.2431C>G; p.L811V are coloboma mutations⁷.</p