A. Primer pair 1 PCR amplification products of FXS negative subjects (lanes 1–5).

<p>Agarose gel electrophoresis results of methylation specific PCR. <b>B.</b> Primer pair 3 gave amplification product of ~80bp of FXS positive subjects (lanes 2–4). <b>C.</b> Primer pair 4 did not give amplification of the FXS positive subjects (lanes 2&3) but yielded a product of ~300bp with FXS negative subjects (lane 4).</p

Molecular Diagnosis of Fragile X Syndrome in Subjects with Intellectual Disability of Unknown Origin: Implications of Its Prevalence in Regional Pakistan

<div><p>Fragile-X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and affects 0.7–3.0% of intellectually compromised population of unknown etiology worldwide. It is mostly caused by repeat expansion mutations in the <i>FMR1</i> at chromosome Xq27.3. The present study aimed to develop molecular diagnostic tools for a better detection of FXS, to assess implementation of diagnostic protocols in a developing country and to estimate the prevalence of FXS in a cohort of intellectually disabled subjects from Pakistan. From a large pool of individuals with below normal IQ range, 395 subjects with intellectual disability of unknown etiology belonging to different regions of the country were recruited. Conventional-PCR, modified-PCR and Southern blot analysis methods were employed for the detection of CGG repeat polymorphisms in the <i>FMR1</i> gene. Initial screening with conventional-PCR identified 13 suspected patients. Subsequent investigations through modified PCR and Southern blot analyses confirmed the presence of the <i>FMR1</i> mutation, suggesting a prevalence of 3.5% and 2.8% (mean 3.3%) among the male and female ID patients, respectively. These diagnostic methods were further customized with the in-house conditions to offer robust screening of referral patients/families for diagnostics and genetic counseling. Prescreening and early diagnosis are crucial for designing a prudent strategy for the management of subjects with ID. Outcome of the study recommends health practitioners for implementation of molecular based FXS diagnosis in routine clinical practice to give a better care for patients similar to the ones included in the study.</p></div

Figure 5

<p>Deletion Analysis Reveals a Critical Role of <i>hc-CNE1-125bp</i> for the Regulatory Potential of CNE1. (A) BLASTZ alignment of a human, mouse, chick, frog, and <i>Fugu</i> highly-conserved 125 bp sequence fragment embedded within CNE1 shown with predicted conserved TFBSs (above). (B) SLAGAN alignment plots of human, mouse, chick, frog and <i>Fugu</i> CNE1 using human sequence as the base line. (C) Architecture of CNE1 wild type and deletion constructs, The red bar depicts the highly conserved region, and less well conserved regions are shown in black. Luciferase activity obtained in H661 cells after transient transfection of reporter constructs is shown in the diagram at the right side. Reporter gene expression is driven by CNE1 fragments upstream of the human <i>GLI3</i> minimal promoter. The red bar depicts luciferase expression (100%) in H661 cells driven alone by the control <i>GLI3</i> minimal promoter (Prom-<i>GLI3</i>-300), while green bars represent the activity recorded for the vectors containing experimental reporter constructs.</p

Figure 4

<p>Tissue Type Specific Expression of GFP Reporter Gene in Zebrafish Embryos. Examples of GFP expression induced by CNEs 1, 9, 10, and 11 are shown in fixed tissues after wholemount anti-GFP immunostaining (bright field views A and F) or in live embryos by combined bright field and GFP fluorescence microscopy analyses (B, C, D, E, G and H). Arrowheads indicate GFP expressing cells. Embryos C and D are ∼26–33 hpf, while embryos A, B, E, F, G, and H are 48–54 hpf. Lateral views, anterior to the left and dorsal to the top except for F where the dorsal view is shown. GFP positive cells were found in the following: (A) CNE1, heart chamber (B) CNE1, hindbrain neurons (C) CNE9, notochord (D) CNE9, spinal cord neuron (E) CNE10, lower jaw primordia and pericardial regions (F) CNE10, lens epithelial cell layer (G) CNE11, pectoral fin (H) CNE11, muscle. (e) Eye; (f) fin; (h) heart; (hb) hindbrain; (I) lens; (nc) notochord; (ov) otic vesicle; (r) retina; (s) spinal cord; (y) yolk.</p

Tetrapod-Teleost Conserved Non-Coding elements (CNEs) from Introns of Human <i>GLI3</i> Selected for Functional Analysis

<p>Location, size, coordinates (NCBI 36, Oct 2005), and human-<i>Fugu</i> conserved transcriptional factor binding sites (union of results from rVISTA and ConSite) are indicated. Dual nature and repressor elements are represented by “A/R” (activator/repressor) and “R” symbols, respectively. The (+) sign indicates the elements which induced GFP expression in zebrafish embryos, while (−) sign indicates those which could not drive GFP expression significantly. n.a.: not analyzed. The analysis of CNE2 is reported elsewhere.</p

Selected pedigrees of FXS patients.

<p>Analyzed patients and carrier mothers are given identification numbers.</p

Figure 1

<p>Comparative Sequence Analysis of the <i>GLI3</i> Locus Detects Conserved Non-coding Sequence Elements. (A) Sequence alignments of the genomic interval containing the human <i>GLI3</i> locus and flanking human genes <i>INHBA</i> and <i>PSMA2</i> with orthologous counterparts from representative members of rodent, bird, amphibian, and fish lineages. These are shown as SLAGAN derived VISTA representations. Conserved coding sequences are depicted in blue and conserved non-coding sequences are in pink. Criteria of alignment were 60 bp window and 50% conservation cutoff. Conservation between human and <i>Fugu</i> (scaffold_210 ENSEMBL genome browser) is restricted to the <i>GLI3</i> gene. Red bars above the conservation plot depict the approximate length of intergenic regions flanking human <i>GLI3.</i> The blue arrow shows the length of the <i>GLI3</i> gene and the direction of transcription. A graphic representation showing exons and introns of <i>GLI3</i> is shown below the homology plot. Green vertical lines indicate the positions of alterations affecting the genomic structure of the locus which result in loss of GLI3 function: a translocation event associated with Greig cephalopolysyndactyly syndrome (GCPS) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000366#pone.0000366-Kruger1" target="_blank">[36]</a>, and two insertions (ins) in mouse mutants anterior digit pattern deformity (<i>add</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000366#pone.0000366-Pohl1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000366#pone.0000366-vanderHoeven1" target="_blank">[37]</a> and polydactyly Nagoya (<i>Pdn</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000366#pone.0000366-Ueta1" target="_blank">[38]</a>. (B) Magnified view of the human/<i>Fugu</i> conservation plot and the genomic structure of human <i>GLI3</i>. The red vertical bars below the plot show the position of human/<i>Fugu</i> highly conserved non-coding sequence elements (CNEs) that were functionally tested as putative enhancers.</p

Figure 7

<p>CNE6 Sequences Flanking Human/Fish conserved Track Show Residual Enhancer Activity. (A) BLASTZ alignment of the highest conserved 35 bp along with two predicted conserved TFBSs from the human/<i>Fugu</i> conserved block within CNE6. (B) CNE6 alignment plot of human, mouse, chick, frog and <i>Fugu</i> sequences using human sequence as the base line. (C) Architecture of wild type and deletion constructs; the red bar depicts the highly conserved human/fish segment. Luciferase activity obtained in H661 cells after transient transfection of reporter constructs is shown in the diagram at the right side. Reporter gene expression is driven by CNE5 fragments upstream of the human <i>GLI3</i> minimal promoter. The red bar depicts luciferase expression (100%) in H661 cells driven alone by the control <i>GLI3</i> minimal promoter (Prom<i>GLI3</i>-300), whilst the green bars represent the activity recorded for the vectors containing experimental reporter constructs, i.e. wild type CNE6 (wt 862bp), CNE6 with deleted human/<i>Fugu</i> conserved block (<i>CNE6Δh/f-179bp</i>), and the 72% human/fish conserved fragment (<i>CNE6h/f-179bp</i>). <i>CNE6Δh/f-179bp</i> can still enhance reporter gene transcription more than two-fold. The isolated 179 bp fragment cannot activate expression.</p

Southern blot analysis shows a normal female control (lane 2); premutation carrier females (lanes 3 and 7); methylation mosaic male (lane 4); full mutation female (lane 5); size mosaic males (lanes 6 and 8).

<p>1kb DNA size marker is shown in lane 1.</p

Facial features of unrelated Fragile X patients.

<p>Facial features of unrelated Fragile X patients.</p