The breakpoints of disease-associated interchromosomal insertions at Xq27.1 localize near the center of 180 bp palindrome sequence.

<p>Cartoon depicts a portion of the palindrome sequence (chrX:139,502,939–139,502,970) with the positive strand folded upon itself in a hairpin loop (black). The four non-palindromic bases in the middle of the 180 bp sequence (TATC, bolded black) are predicted to form the head of the hairpin loop. The locations of the breakpoints on chromosome Xq27.1 for CMTX3 (orange); hypertrichosis¹ (red, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref037" target="_blank">37</a>]); hypertrichosis² (blue, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref037" target="_blank">37</a>]); hypertrichosis³ (green,[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref038" target="_blank">38</a>]); ptosis (pink; Bunyan [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref039" target="_blank">39</a>]); and XX sex reversal (purple, Haines [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref040" target="_blank">40</a>]) are marked out on the hairpin structure. Single breakpoints are depicted by a solid line. Multiple breakpoints are indicated by broken lines.</p

Average depth ± SD of sequence coverage across 8q24.3.

<p>Average depth ± SD of sequence coverage across 8q24.3.</p

Characterization of the CMTX3 insertion.

<p>Sequence analysis of the proximal (A) and distal (B) breakpoints. Reference sequence for chromosome X and chromosome 8 are indicated in blue and orange, respectively. The distal breakpoint includes additional sequence from chromosome 12 (in green) and small rearrangements of the chromosome X sequence including an inversion of 12 bp, and a base pair substitution and a base pair deletion. (C) The 78 kb 8q24.3 sequence (in orange) contains the partial 5’<i>ARHGAP39</i> transcript which has been inserted 330 kb downstream and 84 kb upstream of the genes <i>LOC389895</i> and <i>SOX3</i>, respectively (in blue). The direction of transcripts are indicated by the arrow. (D) Location of the 78kb 8q24.3 insertion sequence (in orange) relative to the whole of the 5.7 Mb CMTX3 locus (in blue).</p

Identification and confirmation of a 78 kb chromosome 8q24.3 insertion in patients with CMTX3.

<p>(A) Whole genome sequencing depth of coverage for affected (red) and normal (black) males across the 8q24.3 insertion and flanking sequence. (B) Depiction of wild type chromosome X (top) and mutant chromosome X (bottom). The location of primers and amplicon sizes for the multiplex PCR genotyping assay are shown. Dotted red lines represent insertion breakpoints. (C) Size fractionation of multiplex PCR genotyping assay for a subset of family members from CMT623. Individual genotypes are depicted above the gel lane. Expected band sizes for the various primer combinations are listed to the right. Unaffected hemizygous males and homozygous females generate a single 340 bp amplicon; affected hemizygous males generate 595 bp and 235 bp amplicons crossing the proximal and distal breakpoints, respectively; carrier females amplify all three amplicons.</p

Split-reads and discordant paired ends mapping to Xq27.1 and 8q24.3 in whole genome sequencing data from affected males.

<p>Split-reads and discordant paired ends mapping to Xq27.1 and 8q24.3 in whole genome sequencing data from affected males.</p

Additional file 1: Table S1. of Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort

DSD gene variants. Each variant found in a diagnostic gene (after the filtering and curation process) is shown. In some cases where the gene is inherited in an autosomal recessive manner, two variants are grouped together. Inheritance has been indicated where familial samples were available: negative indicates negative for variant and N/A sample not available. De novo events have only been noted where both parental samples were available and found to be negative for the change. Previously reported refers to a variant being described in either ClinVar, HGMD, or a publication in a peer-reviewed journal via a PubMed search. Variants were classified consistent with previous MPS publications of DSD cohorts [8, 10] which were based on ACMG guidelines [15]. VUS were called for three reasons: 1 = fits phenotype but predicted to be benign; 2 = damaging but doesn’t fit phenotype; or 3 = variant in the AR repetitive region. Patients marked with an asterisk were identified to have two or more diagnostic gene variants. Null variants (frameshifts, splice sites mutations, and premature stop codons) are shown in bold. Patients have been classified based on clinical notes provided, according to the recommended classification of DSD in the Chicago consensus report. Classifications: CGD complete gonadal dysgenesis, DASA disorders of androgen synthesis or action, DSD DSD of “unknown” origin; hypospadias, LCH Leydig cell hypoplasia, OT ovotesticular DSD, PGD partial gonadal dysgenesis, PMDS persistent Müllerian duct syndrome; syndromic, T testicular DSD. Related affected individuals are indicated. File is in Excel spreadsheet format. (XLSX 47 kb