Primers used in the Sequenom study to amplify <i>IGF2</i>, <i>ICR1/H19</i> and <i>GRB10</i> regions.

<p>Primers used in the Sequenom study to amplify <i>IGF2</i>, <i>ICR1/H19</i> and <i>GRB10</i> regions.</p

XLalphaS and Gsalpha expression in platelets from AHO-like patients.

<p>XLalphaS, CAP1 and Gsalpha expression in AHO-like platelets. A. Immunoblot analysis of XLalphas, CAP1 and Gsalpha protein in platelet lysates from XL hypermethylated AHO-like patients 12, 13, 10, 14, 15, 16 and 3 controls and B. correspondent densitometric scanning of XLalphaS protein in platelet lysates from AHO-like patients with XL hypermethylation (patients 6 to 16) and 5 controls (Controls). Results are expressed as percentage of controls (taken as 100%). Mean as well as SD are depicted as black horizontal and vertical lines, respectively. *, p value<0.05, two-tailed unpaired T-test.</p

Overall <i>GNAS</i>, <i>IGF2</i>, <i>H19</i>, <i>SNURF</i> and <i>GRB10</i> methylation in AHO-like patients.

<p>Dot plot representation of overall methylation values (averages expressed as % of methylation) for NESP, XL, Exon A/B (A), <i>IGF2</i>, <i>H19</i> (B), <i>SNURF</i> and <i>GRB10</i> (C) in AHO-like patients (indicated as ‘PPHP’) vs. the control population (indicated as ‘crls’). Individuals with significant hyper- or hypomethylation (patients 1 to 5) in the NESP, Exon A/B, H19 and GRB10 are indicated as follow: patient 1 =  red, 2 =  green, 3 =  blue, 4 =  brown, 5 =  yellow. Medians are displayed as black lines. ** p<0.01 and * p<0.05, two-tailed unpaired T-test.</p

Clinical patients' characteristics and platelet Gs activity.

*<p>Small-for-Gestational-Age (SGA); ∧Gilles de la Tourette; °ADHD; SSC: subcutaneous calcifications; Ob: obesity; RF: round face; ID: Intellectual disability; NA: not available.</p>**<p>vs. crls, p<0.05 The concentration of Gsα agonist to inhibit the collagen-induced platelet aggregation by 50 % (IC₅₀) is indicated between brackets for 24 normal controls. A Gsα hypofunction is defined as requiring a significantly higher IC₅₀ value.</p

Chromosomal location of the <i>IGF2</i>, <i>H19</i> and <i>GRB10</i> amplicons used in the Sequenom study.

*<p>Nucleotide positions according to the February 2009 human reference sequence (GRCh37/hg19) produced by the International Human Genome Sequencing Consortium.</p

Phenotypic, Molecular Genetic and Platelet Gs protein activity in relation to <i>GNAS</i> pathology.

<p>Phenotypic, Molecular Genetic and Platelet Gs protein activity in relation to <i>GNAS</i> pathology.</p

Pseudoautosomal Region 1 Length Polymorphism in the Human Population

<div><p>The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into theMa Y chromosome generates an extended PAR. The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.</p></div

Reciprocal deletion.

<p>(A) 180K Custom Microarray aCGH results with the upper track displaying an overview of the log2-ratio of the fluorescent signal across the entire X chromosome and the central track displaying a zoomed in portion of the X chromosome, with the log2-ratio of the fluorescent signals on the Y Axis. (B) PCR bands across the deletion region for the deletion carrying patient (P), father (F), mother (M), sister (S), female control (fc), male control (mc), and negative control (neg). (C) Sequencing of the amplicon in part B for the patient. Red letters indicate X specific reference sequence, yellow letters indicate LTR6B reference sequence, yellow letters highlighted in red indicate sequence specific for X specific LTR6B, yellow letters highlighted in purple indicate sequence specific for pseudoautosomal LTR6B, purple letters highlighted in purple indicate sequence originated from PAR1 that was not sequenced but contains the forward primer site, and yellow letters highlighted in yellow indicate LTR6B sequence that was not sequenced but contains the forward primer site. Highlighted in black are annotated SNPs.</p

Sequencing to validate the insertion and demonstrate recurrence.

<p>(A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.</p

Identification of an X specific insertional translocation in Y.

<p>(A) Determining a duplication by array-CGH using a 180K Custom Microarray. The upper track shows an overview of the log2-ratio of the fluorescent signal across the entire X chromosome. The central track shows a zoomed in portion of the X chromosome, with the log2-ratio of the fluorescent signals on the Y Axis. (B) Further zooming in on the duplication, including the location of genes <i>XG</i> and <i>GYG2</i>. (C) FISH results for a father carrier (1, 3) and male control (2, 4) using probes targeting PAR1 (1, 2) and the duplicated region (3, 4). Note the presence of PAR1 on X and Y chromosomes in the carrier father and control, the PAR flanking probe on the X chromosome in both individuals, but the X insertion signal is found only on the Y chromosome of the carrier father. (D) The heterozygous SNP profile from Illumina sequencing of patient P1 for hg19 chrX:2,680,000-2,830,000. The dashed vertical gray line indicates the pseudoautosomal boundary, the yellow vertical lines illustrate LTR6B positions, gray diamonds illustrate heterozygote SNPs, and the black horizontal lines indicate mean frequencies of all depicted SNPs in the span of the line. Across the top is illustrated chromosome X, with unique X sequence in red, PAR1 reference sequence in purple, and LTR6B's in yellow. Note the presence of heterozygote SNPs in the X specific region, and the SNPs featuring frequencies of 0.33 in the proximal PAR1.</p