Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes

International audiencehe frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability 'homemade' make it a valuable tool as a new diagnostic approach of CNMs

Loss-of-function mutations in SOX10 cause Kallmann syndrome with deafness.

International audienceTranscription factor SOX10 plays a role in the maintenance of progenitor cell multipotency, lineage specification, and cell differentiation and is a major actor in the development of the neural crest. It has been implicated in Waardenburg syndrome (WS), a rare disorder characterized by the association between pigmentation abnormalities and deafness, but SOX10 mutations cause a variable phenotype that spreads over the initial limits of the syndrome definition. On the basis of recent findings of olfactory-bulb agenesis in WS individuals, we suspected SOX10 was also involved in Kallmann syndrome (KS). KS is defined by the association between anosmia and hypogonadotropic hypogonadism due to incomplete migration of neuroendocrine gonadotropin-releasing hormone (GnRH) cells along the olfactory, vomeronasal, and terminal nerves. Mutations in any of the nine genes identified to date account for only 30% of the KS cases. KS can be either isolated or associated with a variety of other symptoms, including deafness. This study reports SOX10 loss-of-function mutations in approximately one-third of KS individuals with deafness, indicating a substantial involvement in this clinical condition. Study of SOX10-null mutant mice revealed a developmental role of SOX10 in a subpopulation of glial cells called olfactory ensheathing cells. These mice indeed showed an almost complete absence of these cells along the olfactory nerve pathway, as well as defasciculation and misrouting of the nerve fibers, impaired migration of GnRH cells, and disorganization of the olfactory nerve layer of the olfactory bulbs

The prevalence of CHD7 missense versus truncating mutations is higher in patients with Kallmann syndrome than in typical CHARGE patients

International audienceCONTEXT: Mutations in CHD7, a gene previously implicated in CHARGE (coloboma, heart defect, choanal atresia, retardation of growth and/or development, genital hypoplasia, ear anomalies) syndrome, have been reported in patients presenting with Kallmann syndrome (KS) or congenital hypogonadotropic hypogonadism (CHH). Most mutations causing CHARGE syndrome result in premature stop codons and occur de novo, but the proportion of truncating vs nontruncating mutations in KS and CHH patients is still unknown. OBJECTIVE: The objective of the study was to determine the nature, prevalence, mode of transmission, and clinical spectrum of CHD7 mutations in a large series of patients. DESIGN: We studied 209 KS and 94 CHH patients. These patients had not been diagnosed with CHARGE syndrome according to the current criteria. We searched for mutations in 16 KS and CHH genes including CHD7. RESULTS: We found presumably pathogenic mutations in CHD7 in 24 KS patients but not in CHH patients. Nontruncating mutations (16 missense and a two-codon duplication) were more prevalent than truncating mutations (three nonsense, three frame shift, and a splice site), which contrasts with patients presenting with typical CHARGE syndrome. Thus, the clinical spectrum associated with CHD7 mutations may be partly explained by genotype/phenotype correlations. Eight patients also had congenital deafness and one had a cleft lip/palate, whereas six had both. For 10 patients, the presence of diverse features of the CHARGE spectrum in at least one relative argues against a de novo appearance of the missense mutation, and this was confirmed by genetic analysis in five families. CONCLUSION: Considering the large prevalence and clinical spectrum of CHD7 mutations, it will be particularly relevant to genetic counseling to search for mutations in this gene in KS patients seeking fertility treatment, especially if KS is associated with deafness and cleft lip/palate.</p

SEMA3A, a Gene Involved in Axonal Pathfinding, Is Mutated in Patients with Kallmann Syndrome

International audienceKallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder

Expression of the Sema3A coreceptor Nrp1 by vomeronasal/terminal nerve fibers and migrating GnRH cells in human and mouse embryos.

<p>(A) Schematic representation of the head of a mouse embryo at E14.5, showing the scaffold of vomeronasal/terminal nerve fibers (in red) along which GnRH cells (in blue) migrate from the nose to the ventral forebrain region. Several areas along this migratory path have been shown to produce Sema3A, including the frontonasal mesenchyme and the olfactory bulb region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002896#pgen.1002896-Schwarting1" target="_blank">[21]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002896#pgen.1002896-Giger1" target="_blank">[33]</a>. Boxes indicate the locations of the sagittal sections shown in (B) and (C). Abbreviations: oe, olfactory epithelium; vno, vomeronasal organ; nm, frontonasal mesenchyme; mob, main olfactory bulb; aob, accessory olfactory bulb; vfb, ventral forebrain; 3V, third ventricle. (B) Sagittal section of the frontonasal region in an E14.5 mouse embryo. In the frontonasal mesenchyme (nm), migrating GnRH-immunoreactive cells (green) are morphologically associated with Nrp1-immunoreactive nerve fibers (red) originating in the vomeronasal organ (vno). Single plane confocal images at higher magnification (insets) show that GnRH cells are Nrp1-immunoreactive (green+red = yellow staining). (C) Sagittal section of the ventral forebrain (vfb) in an E14.5 mouse embryo. The peripherin-immunoreactive (green) fibers of the caudal branch of the vomeronasal nerve (arrows) are also Nrp1-immunoreactive (red), as shown by their yellow staining (green+red). (D) Sagittal section of the olfactory epithelium (oe) and olfactory bulb (ob) regions (left panel) and detail of the frontonasal region (right panel) in a 9 week-old human fetus. Clusters of GnRH-immunoreactive cells (green, arrowheads) are visible in the frontonasal mesenchyme (nm) and the rostral forebrain (fb). In the frontonasal region, these cells migrate in close contact with Nrp1-immunoreactive axons (red). Note that migrating GnRH cells are also Nrp1-immunoreactive, as shown by their yellow staining (green+red) in the right panel (arrows). Scale bars: 100 µm (25 µm in insets).</p

<i>SEMA3A</i> mutations identified in Kallmann syndrome patients.

<p><i>SEMA3A</i> mutations identified in Kallmann syndrome patients.</p

Defects in olfactory and vomeronasal axons, and GnRH cell migration in <i>Nrp1</i>^sema/sema mutant mice.

<p>(A) Coronal sections of the right olfactory epithelium (oe) and olfactory bulb (ob) regions (left panels), and detail of the olfactory bulb showing the olfactory nerve layer (nl) and glomerular layer (gl) (right panels) in <i>Nrp1</i>^+/+ and <i>Nrp1</i>^sema/sema newborn (P0) mice. Axons of the olfactory receptor neurons were immunostained (red) using an antibody directed against the olfactory marker protein (OMP). In the <i>Nrp1</i>^sema/sema mouse, the immunostaining is both enlarged below the olfactory bulb ventro-medial aspect (asterisks) and markedly reduced in the glomerular layer (arrowheads) compared to wild-type. (B) Sagittal sections of the rostral and ventral forebrain regions (left panels), and detail of the caudal branch of the vomeronasal nerve (right panels) in <i>Nrp1</i>^+/+ and <i>Nrp1</i>^sema/sema E14.5 mouse embryos. A crystal of the DiI lipophilic fluorescent dye has been placed in the vomeronasal organ lumen to anterogradely label vomeronasal axons. The vomeronasal nerve extends across the medial aspect of the olfactory bulb and projects both dorsally, to the accessory olfactory bulb, and caudally, to the ventral forebrain (vfb). In the mutant mouse, fibers in the caudal branch are scarce compared to wild-type. (C) Sagittal sections of the rostral and ventral forebrain regions at E14.5, immunostained for GnRH (green). Note the abnormal distribution of GnRH-immunoreactive cells in the <i>Nrp1</i>^sema/sema mouse (arrows). (D) Coronal sections of the preoptic region (upper panels) showing GnRH neuroendocrine cells (green) and their projections in the median eminence (me, arrows) (lower panels) in <i>Nrp1</i>^+/+ and <i>Nrp1</i>^sema/sema newborn (P0) mice. The immunostaining is reduced in the <i>Nrp1</i>^sema/sema mouse. (E) Quantitative analysis (mean ± s.d.) of GnRH cell distributions in <i>Nrp1</i>^+/+ and <i>Nrp1</i>^sema/sema mice at E14.5 and P0. * and ** denote statistically significant differences between genotypes in the indicated head regions (two-way ANOVA followed by Tukey's range test) with <i>p</i><0.05 and <i>p</i><0.01, respectively. Note that the total numbers of GnRH cells are not statistically different between <i>Nrp1</i>^+/+ and <i>Nrp1</i>^sema/sema mice at E14.5 or P0 (Student's t-test, <i>p</i>>0.05). Other abbreviations: cx, cerebral cortex; ovlt, organum vasculosum of lamina terminalis; 3v, third ventricle. Scale bars: 100 µm (50 µm in inset).</p