Diagnostic value of exome and whole genome sequencing in craniosynostosis

Background Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in ~1 in 2250 births, either in isolation or as part of a syndrome. Mutations in at least 57 genes have been associated with craniosynostosis, but only a minority of these are included in routine laboratory genetic testing. Methods We used exome or whole genome sequencing to seek a genetic cause in a cohort of 40 subjects with craniosynostosis, selected by clinical or molecular geneticists as being high-priority cases, and in whom prior clinically driven genetic testing had been negative. Results We identified likely associated mutations in 15 patients (37.5%), involving 14 different genes. All genes were mutated in single families, except for IL11RA (two families). We classified the other positive diagnoses as follows: commonly mutated craniosynostosis genes with atypical presentation (EFNB1, TWIST1); other core craniosynostosis genes (CDC45, MSX2, ZIC1); genes for which mutations are only rarely associated with craniosynostosis (FBN1, HUWE1, KRAS, STAT3); and known disease genes for which a causal relationship with craniosynostosis is currently unknown (AHDC1, NTRK2). In two further families, likely novel disease genes are currently undergoing functional validation. In 5 of the 15 positive cases, the (previously unanticipated) molecular diagnosis had immediate, actionable consequences for either genetic or medical management (mutations in EFNB1, FBN1, KRAS, NTRK2, STAT3). Conclusions This substantial genetic heterogeneity, and the multiple actionable mutations identified, emphasises the benefits of exome/whole genome sequencing to identify causal mutations in craniosynostosis cases for which routine clinical testing has yielded negative results

Missense variants in ANKRD11 cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein

Purpose Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants. Methods We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants through in silico analyses and cell-based experiments. Results We identified 20 unique, mostly de novo, ANKRD11 missense variants in 29 individuals, presenting with syndromic neurodevelopmental disorders similar to KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in repression domain 2 at the ANKRD11 C-terminus. Of the 10 functionally studied missense variants, 6 reduced ANKRD11 stability. One variant caused decreased proteasome degradation and loss of ANKRD11 transcriptional activity. Conclusion Our study indicates that pathogenic heterozygous ANKRD11 missense variants cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges because the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping

Distribution of Glycine/GABA Neurons in the Ventromedial Medulla with Descending Spinal Projections and Evidence for an Ascending Glycine/GABA Projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2+GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29%61 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31%63 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25%62 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spina

Neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNAs.

<p>Fluorescence micrographs (A1, B1) and their magnifications (A2, B2) showing neurons in the rostral ventromedial medulla (RVM) retrogradely labeled with fluorescent microspheres (red) from the lumbar (L4) dorsal horn (<b>A</b>) and the cervical (C5) spinal cord (<b>B</b>), and neurons labeled for GlyT2 and/or GAD67 mRNAs (Gly/GABA neurons, green) using fluorescent in situ hybridization (FISH). Arrowheads indicate Gly/GABA neurons that are also retrogradely labeled from the spinal cord. Note that the fluorescent microspheres and the labeling for Gly/GAD67 mRNA are located in the cytoplasm, outside of the neuronal nuclei. The intensity of the fluorescent labeling varied between the labeled neurons, several of which are also out of focus. Gi: gigantocellular reticular nucleus, alpha part (GiA); LPGi: lateral paragigantocellular reticular nucleus, alpha part (LPGiA), external part (LPGiE); Ml: medial lemniscus; RMg: raphe magnus nucleus. Scale bar: 50 µm.</p

The injection sites of fluorescent microspheres in the rat spinal cord.

<p>Drawings illustrating fluorescent microsphere injection sites in the dorsal and ventral cervical (C5–C6) spinal cord (<b>A</b>), in the dorsal horn (laminae I to VI) (<b>B</b>) and in the ventral horn (laminae VII-X) (<b>C</b>) of the lumbar (L4–L6) spinal cord. Note that all injections were confined to one side, and that in some cases fluorescent tracer was also present the adjacent white matter. Roman figures indicate spinal laminae.</p

Spinal neurons retrogradely labeled from the rostral ventromedial medulla, and spinal neurons labeled for GlyT2 and/or GAD67 mRNAs.

<p>Fluorescence micrographs showing spinal neurons (red) retrogradely labeled with fluorescent microspheres from the rostral ventromedial medulla (RVM) and spinal neurons (green) containing GlyT2 and/or GAD67 mRNA (glycinergic and/or GABAergic neurons) identified with FISH. Arrowheads indicate spinal neurons in the area around the central canal, which are retrogradely labeled from the RVM and are also labeled for GlyT2 mRNA in segment C5 (<b>A</b>) and for GlyT2 and/or GAD67 mRNAs in segments L5 (C) and L6 (<b>B and D</b>). Scale bar = 50 µm.</p

Neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNAs.

<p>Fluorescence micrographs showing neurons in the ventromedial medulla retrogradely labeled with fluorescent microspheres (red) from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNA (green) using FISH. Arrowheads indicate GABAergic (GAD67 mRNA containing) neurons in the caudal ventromedial medulla (CVM) retrogradely labeled from the cervical spinal cord (<b>A</b>); glycinergic (GlyT2 mRNA containing) neurons in the rostral ventromedial medulla (RVM) retrogradely labeled from the cervical spinal cord (<b>B</b>); GlyT2 and/or GAD67 mRNA containing neurons (Gly/GABA) in the RVM retrogradely labeled from the lumbar dorsal horn (<b>C</b>); Gly/GABA neurons in the RVM retrogradely labeled from the lumbar ventral horn (<b>D</b>); Gly/GABA neurons in the CVM retrogradely labeled from the lumbar ventral horn (<b>E</b>). Scale bar = 50 µm.</p

The distribution patterns of neurons in the spinal cord that were retrogradely labeled from the rostral ventromedial medulla.

<p>Schematic drawings showing the brainstems of three rats with fluorescent microsphere injections (blue, purple and red) and the corresponding spinal cords with the distribution of retrogradely labeled neurons (filled black circles), and retrogradely labeled spinal neurons also containing GlyT2 and/or GAD67 mRNA (red triangles). Each segmental drawing, i.e. cervical (C4–C8), thoracic (T1–T3 and T10–T12), lumbar (L1–L6) and sacral (S1–S4), shows the cumulative results obtained in 8-10 sections. Tracer injections were aimed at rostral ventromedial medulla (RVM) with injections confined to the midline (purple), to one side only (blue), or both sides of the RVM (red). Note that tracer injections limited to the midline yield fewer retrogradely labeled neurons in the spinal cord (purple), and injections limited to one side of the brainstem, yield more retrogradely labeled neurons on the contralateral side of the spinal cord (blue as compared to purple and red). Roman figures indicate spinal laminae. LSN: lateral spinal nucleus.</p

The distribution patterns of neurons in the ventromedial medulla that were retrogradely labeled from the lumbar dorsal or ventral horn.

<p>Drawings illustrating representative single brainstem sections of the rostral part of ventromedial medulla (RVM) and the caudal part of ventromedial medulla (CVM). Black filled circles represent neurons retrogradely labeled with fluorescent microspheres from the lumbar dorsal (<b>A</b>) or ventral (<b>B</b>) horn. Red filled triangles represent retrogradely labeled neurons that also contained GlyT2 and/or GAD67 mRNA (Gly/GABA). Note that the caudal part of the CVM contains fewer retrogradely labeled neurons than the rostral part of the CVM, and that most retrogradely labeled Gly/GABA neurons in both the CVM and RVM are located on the side ipsilateral to the injection. Gi: gigantocellular reticular nucleus, alpha part (GiA); IRt: intermediate reticular nucleus, alpha part (IRtA); LPGi: lateral paragigantocellular reticular nucleus, alpha part (LPGiA), external part (LPGiE); LRt: lateral reticular nucleus; Pn: pontine reticular nucleus, ventral part (PnV) and caudal part (PnC); Py: pyramid tract; Oi: olivary inferior nucleus; RMg: raphe magnus; Tz: nucleus trapezoid body; 7Nuc: nucleus of seventh nerve; 1: raphe pallidus nucleus; 2: raphe obscurus nucleus; 3: raphe interpositus nucleus.</p