Amplification and dHPLC conditions for <i>PHOX2B</i> genomic sequence analysis.

<p>Bp: size of the fragment; B: buffer concentration; CM: MgCl2 concentration; DNTPs: dNTPs concentration; P: primers concentration; Taq: units of Taq polymerase; DNA: DNA quantity; D: Denaturing temperature; DT: Denaturing time; H: Hybridization temperature; HT: Hybridization time; E: Extension temperature; ET: Extension time; T dHPLC: Injection temperature in dHPLC. The general PCR protocol was [95°C-5′]→[(D–DT) →(H–HT) → (E–ET)]×35cycles→[72°C–7′]→4°C.</p

<i>PHOX2B</i> deletion detected in exon 2.

<p>(A) Schematic representation of <i>PHOX2B</i> showing both nucleotide and amino acid sequence of exon 2; the 18bp corresponding to the deletion are boxed. (B) Sequence chromatogram of <i>PHOX2B</i> exon 2; the arrow indicates the beginning of c.393_410del18bp. (C) Pedigree and agarose gel electrophoresis of the amplification product of exon 2 in the patient and her parents.</p

Structure of the homeodomain.

<p>(A) Multiple sequence alignment among different species. Aminoacid residues involved in the deletion are included within a red box. (B) Three-dimensional representation of the interaction between the <i>Drosophila sp.</i> homeodomain and the target DNA sequence (orange). Aminoacids encompassed by the deletion are represented in green. C) Prediction of the electrostatic potential for the wild type protein (up) and the mutated one (down). Negative charge is represented in blue and positive charge in red.</p

Additional file 1: of Next generation phenotyping using narrative reports in a rare disease clinical data warehouse

Extracted phenotypical concepts per cohort. For each cohort, we list the top50 concepts ranked by Frequency and TF-IDF. The first column is the UMLS code of the phenotypical concepts, the second column is the French preferred terms, the third column is the English preferred terms, the fourth column is the frequencies score (FREQ), the fifth column is the TF-IDF score, the sixth column is the rank of the concept sorted by the frequency score, the seventh column is the rank of the concept sorted by the TF-IDF score and the eighth column is the expert evaluation (1: relevant concept, 0: none relevant concept). (XLS 93 kb

Dual-fluorescence transgenic analysis of a SHH enhancer SBE2 where a C>T change in a Six3 binding site has been identified in a patient with holoprosencephaly.

<p><b>(A)</b> Regulatory landscape of SHH, depicting the location of the SBE2 enhancer. The sequence conservation plot on the right shows the low level of sequence conservation for SBE2 in the zebrafish genome. <b>(B)</b> SHH-SBE2 enhancer-driven F1 reporter transgenics. The wild-type allele Wt(C) drives expression in the rostral and caudal hypothalamus (long green or red bar) independent of the associated fluorophore (eGFP or mCherry). Expression from the mutant allele, Mut(T), is retained in the caudal hypothalamus (short green or red bar) but lost in the rostral hypothalamus (short green or red bar). Additional expression of the SBE2 Wt(C) allele in the rostral forebrain of later stage (96hpf) zebrafish embryos is also lost by the Mut(T) allele. <b>(C)</b> RNA <i>in situ</i> hybridisation analysis of <i>shha</i> and <i>shhb</i> expression at 48hpf and 96hpf of zebrafish embryonic development. The reporter gene expression pattern driven by Wt(C) allele significantly overlaps with the <i>shha</i> expression domain in the hypothalamus. <b>(D)</b> Morpholino knock-down of Six3 in SHH-SBE2 transgenic embryos mimics the effect of the Six3 binding site mutation in SBE2. F2 embryos bearing both SHH-SBE2 Wt(C) and Mut(T) alleles injected with either control morpholino (Control MO) or morpholino against both six3A and six3B (six3 AMO). Upon knockdown of six3, the hypothalamus expression driven by the Wt allele shrinks to overlap completely with the expression driven by the mutant allele (bearing a mutant six3 binding site). Wt: wild-type; Mut: mutant; hpf: hours post fertilization; MO: morpholino, RH: Rostral hypothalamus; CH: Caudal hypothalamus; rFB: Rostral forebrain.</p

Novel PRS-associated elements tested for enhancer function.

<p>Novel PRS-associated elements tested for enhancer function.</p

Known disease-associated CRE variants studied.

<p>Known disease-associated CRE variants studied.</p

Dual-fluorescence transgenic analysis of established CREs with disease-associated point mutations.

<p><b>(A)</b> SHH-ZRS. (a) The regulatory landscape of SHH, depicting the location of ZRS enhancer. The conservation plot on the left shows the low level of sequence conservation for ZRS in the zebrafish genome. (b) SHH-ZRS enhancer-driven reporter expression is shown at 72 hpf and 96 hpf. The top row shows the Wt(G) allele driving both mCherry and eGFP expression to equal measure in a restricted region of the developing pectoral fin (PF), coinciding with the expression domain of both <i>shha</i> and <i>shhb</i> in the developing pectoral fin (c). The Mut(A) allele drives expression at an additional site on the opposite edge of the fin. The ectopic expression (arrowheads) is increased by 96 hpf. The double-headed arrow indicates the anterior (a)—posterior (p) axial plane of the developing pectoral fin. <b>(B)</b> PAX6-SIMO. <b>(a)</b> The regulatory landscape of PAX6, depicting the location of SIMO enhancer. The conservation plot on the right shows the presence of sequence conservation for SIMO in the zebrafish genome. (b) PAX6-SIMO enhancer-driven reporter expression is shown at 72 hpf. In contrast to the Wt(G) element, and irrespective of the fluorophore used, the Mut(T) allele consistently fails to drive reporter gene expression in the developing lens (L). (c) RNA <i>in situ</i> hybridisation analysis of zebrafish <i>pax6a</i> at 72hpf showing the endogenous <i>pax6a</i> expression pattern in the developing eye with strong retinal expression and weaker lens expression overlapping with the reporter domain driven by the Wt(G). Wt: wild-type; Mut: mutant; hpf: hours post fertilization.</p

New enhancers from the SOX9 region with PRS-associated single nucleotide variants.

<p><b>(A) p300-PK17</b>, spatiotemporally altered expression: F1 dual-fluorescence reporter transgenic embryos shown at 48, 72 and 96 hpf. At 48 and 72 hpf the Wt(G) allele drives expression in the region around the oral cavity (OC) (white arrowhead). At 96 hpf Wt(G) drives expression in the palatoquadrate (PQ) (curved white arrow) and Meckel’s cartilage (MC) (open arrow). The Mut(A) allele is able to drive OC expression at 48 hpf, but this is lost by 72 hpf and at 96 hpf no PQ and MC expression is observed. <b>(B) hoc-CNE-D</b>, tissue-specific CRE with unaltered expression: F1 dual-fluorescence transgenic embryos at 72 and 96 hpf. The Wt(T) and the Mut(C) alleles both drive expression in olfactory placode (OP), brain (BR), and ceratobranchials (CB). <b>(C) p300-PK22</b>, craniofacial CRE with unaltered expression: F1 dual-fluorescence transgenic embryos at 72 hpf. The Wt(A) and Mut(C) allele both drive expression in the region around the oral cavity (OC). <b>(D-E)</b> RNA <i>in situ</i> hybridisation analysis of zebrafish <i>sox9a</i> and <i>sox9b</i> at 72hpf and 96hpf, showing overlap of reporter gene expression driven by the p300-Pk17, hoc-CNE-D and p300-Pk22 elements with the endogenous <i>sox9a</i> and <i>sox9b</i> expression pattern in the developing jaw. Wt: wild-type; Mut: mutant; hpf: hours post fertilization.</p

Dual-fluorescence transgenic analysis of IRF6 enhancer MCS-9.7 where a SNP (rs642961, G>A) in a TFAP2A (AP2α) binding site has been associated with cleft lip.

<p><b>(A)</b> Regulatory landscape of IRF6, depicting the location of MCS-9.7 enhancer. The conservation plot on the right shows the absence of sequence conservation for MCS-9.7 in the zebrafish genome. <b>(B)</b> IRF6-MCS-9.7 enhancer-driven F1 reporter transgenics. The Wt(G) allele drives expression in the first pharyngeal arch (PA1) (arrow) and in the developing ethmoid plate (EP) (curved arrow). Mut(A) has lost EP expression but maintains PA1 expression. <b>(C)</b> RNA <i>in situ</i> hybridisation analysis of zebrafish <i>irf6</i> at 72hpf showing overlap of the reporter gene expression domain driven by Wt(G) allele with the endogenous <i>irf6</i> expression pattern in the developing jaw. Wt: wild-type; Mut: mutant; hpf: hours post fertilization.</p