Clinical images.

<p>A) Clinical image revealing a whitish-red plaque on the lower right leg of the 4-year-old proband. The affected area is made up of numerous whitish-red round papules that coalesce into irregular plaque and single papules, the area perimeter defined by a whitish border and cleaved by a central furrow. Slightly raised whitish-red portions can also be observed. B) Enlarged detail of the lesion. C) The patient's skin biopsy shows slight papillomatosis and ortokeratosis of epidermis and a cornoid lamella; the derma appears normal (H&E, 10×). D) A column of parakeratotic cells makes up the cornoid lamella (H&E, 200×). E) The examination of the skin biopsy of the father shows atrophic epidermis with two cornoid lamellae; solar elastosis and sparse perivascular lymphocytic infiltrate can be recognized (H&E, 10×). F) The cornoid lamella is very thin (H&E, 200×).</p

Expression studies.

<p>Comparison <i>of EMILIN2, LPIN2</i>, and <i>MAD1L1</i> expression in the primary skin fibroblast cultures of proband's father compared with controls (Ct). The histogram shows the median of values: dark grey corresponds to patient's fibroblasts (#102) normalized to the median of values of six normal control's fibroblasts (light grey).</p

Array-CGH results.

<p>A) Result of array-CGH analysis of chromosome 18 with Agilent Human Genome CGH microarray Kit G3 400K. The 18p11.32p11.31 duplicated region extends between oligomers A_16_P20755613 (2,724,439 bp) and A_16_P40933037 (3,153,981 bp) B) Gene contents of the duplicated region.</p

Clinical signs of the patient 1.

<p>The patient 1 at 3 years of age: short stature, prominent forehead, strabismus, epicanthus, low-set-ears, depressed nasal bridge, short phyltrum, V-shaped mouth. Mild brachydactyly, cutaneous syndactyly of 2nd and 3rd toes.</p

FISH results.

<p>a) FISH with specific subtelomeric 7q probe (red) confirmed the presence of the unbalanced translocation both in the patient and mother. b) FISH with oligonucleotide-based probe 15q13.3 (green signal) and BAC RP11-231A23 (red signal) on the metaphase of the mother in family 1 showing the deletion on a chromosome 15 (arrow). The arrowhead indicates the normal chromosome 15. c) FISH with oligonucleotide-based probe 15q13.3 (green signal) and BAC RP11-231A23 (red signal) on the metaphase of the proband in family 1 showing the duplication on a chromosome 15 (arrow). The arrowhead indicates the normal chromosome 15.FISH performed with the same probes on the metaphase of the mother showed normal signals only on both chromosome 15 (data not showed). d) FISH with oligonucleotide-based probe 22q11.21 (red signal) and BAC RP11-1143M16 (green signal) on the metaphase of the proband in family 2 confirming that large and short deletions are on the same chromosome 22. Arrowhead shows the normal chromosome 22 and arrow the deleted chromosome 22.</p

Clinical signs of the patient 2.

<p>A) Details of feet, showing very long hallux bilaterally B) X-rays, showing severe scoliosis. C) Long and curved lower limbs.</p

Expression and biochemical results.

<p>A) CNP plasma concentration in patient 2, his parents and three controls. The concentration of the peptide was increased more than 4-fold in the propositus compared to parents and unrelated controls (n = 3). B) Analysis of NPPC expression by real-time PCR analysis in lymphoblasts from patient and his parents. The expression of NPPC in the propositus was remarkably higher compared to those of his father and mother.</p

Family 2, array-CGH results.

<p>a) Array-CGH graphical overview of 22q11.21 deletion (large deletion) of the proband. b) Enlargement of the deleted region spanned 1.417 Mb from oligonucleotide A_16_P41477688 (18,894,835 bp) to A_14_P200761(20,311,763 bp). c) Enlargement of the 22q11.23 deleted region (short deletion) spanned 247 Kb from oligonucleotide A_14_P133174 (25,664,618 bp) to A_18_P13974242 (25,911,651 bp) inherited from his father. d) Enlargement of the 15q26.3 duplicated region spanned 484.3 Kb from oligonucleotide A_16_P20368426 (100,051,182 bp) to A_14_P110953 (100,996,155 bp) of the proband and inherited from his father.</p

Expression and biochemical results.

<p>A) CNP plasma concentration in patient 1, his parents and six controls. The concentration of the peptide was normal compared to parents and unrelated controls (n = 6). B) Analysis of <i>NPPC</i> expression by real-time PCR analysis in lymphoblasts from patient, his parents, and ten controls. The expression of NPPC in the proposita was normal compared to those of his parents and unrelated controls (n = 10).</p

Schematic representation of the genomic region spanning the described deletions according to the UCSC Genome Browser Feb 2009 (GRCh37/hg19).

<p>Several annotated genes and ESTs are present, the two deletions are indicated by black arrows (patient 1 and patient 2), whereas triangles indicate the position of the translocation breakpoints previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066048#pone.0066048-Bocciardi1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066048#pone.0066048-Moncla1" target="_blank">[12]</a>.</p