SHH

Research collaboration

Molecular and cellular bases of congenital diseases related to SHH deficiency
Proposed research aims to explain incomplete penetrance and variable expressivity for non-Mendelian rare diseases by characterizing pathogenic combination of rare variants. As number of patients is typically low in rare diseases, identification of such combinatory events requires performing large-scale studies on enlarged disease spectrum.

We have chosen as a model the numerous malformation syndromes caused by a dysfunction of the SHH signaling pathway. SHH operates during early brain development where it induces appropriate dorsoventral patterning of forebrain and eye field. Mutations in SHH itself and its signaling effectors are responsible for different developmental anomalies that cover a broad clinical spectrum of brain malformations. We and others have identified 20 risk genes, which are all implicated in the regulation of SHH dosage during brain development. However, 70 % of SHH-Deficiency (SHH-D) disorder cases still remain unsolved.

Holoprosencephaly (HPE) is the most severe pathology related to a SHH-D and originates from an incomplete cleavage of the forebrain that leads to severe brain and craniofacial anomalies. SHH-D disorders altogether constitute a syndromic continuum ranging from the severe form, HPE, to milder disorders such as malformed pituitary gland, abnormal corpus callosum, microcephaly and coloboma.

Our recent work has demonstrated that oligogenic events accounts for a significant part of HPE cases. Similar to HPE we propose that SHH-D disorders result from combination of variants in genes related to SHH signaling.
In order to test this hypothesis we will:

  • Establish genotype-phenotype correlation through in–depth clinical phenotyping of mildly affected patients from different SHH-D cohorts (HPE, microcephaly, coloboma, etc.).
  • Enrich the list of causative variants and variant combinations by NGS sequencing using our dedicated bioinformatics pipelines.
  • Determine molecular consequences of most relevant variants and their pathogenic combinations on SHH signaling pathway in cellular models.
  • Assess the impact of identified variants and combinations on SHH activity and dorso-ventral brain differentiation using a cerebral organoid model.

Our lab coordinates an international HPE network that is supported by reference centers for rare developmental diseases and that has been extended to additional SHH-D diseases. We have now collected over 2,700 blood samples or frozen fetal tissues of patients and their relatives, from both sporadic and familial cases.

In accordance with oligogenic inheritance, most microforms have been diagnosed because affected individuals were close relatives of patients with severe HPE. Such families obviously represent perfectly appropriate genetic models for our study. We will perform molecular diagnosis and deep phenotyping for all members of families with SHH-D cases, including neuroimaging and determination of clinical traits that are traditionally not considered in the SHH-D spectrum. We expect to provide accurate genotype-phenotype correlation and, thereby, improve diagnosis success rate.

We routinely perform NGS-based analyses to investigate genetic basis of novel HPE patients. We are developing new tools to analyze association of phenotypes with multiple rare variants. Our approach relies on interpreting exome/genome data by combining gene-phenotype associations and external datasets. Successfully applied to HPE, this approach will now be used on an expanded spectrum of disease traits and will provide characterization of spatio-temporal molecular architecture of SHH-D disorders. Exploring medical phenome of SHH-D disorders will also allow deciphering their oligogenic inheritance and addressing if and how SHH-D disorders are modulated by specific variants. Here we anticipate that considering gene regulatory regions in our analysis will shed lights on novel SHH-D related DNA alterations located in the non-coding part of the genome.

Functional analysis of variants/candidate genes will be performed in human cultured cells that are competent for production and excretion of SHH signal on one side and for SHH-dependent cell signaling down to target gene expression on the other side. In a first approach, we will determine actual implication of variants/candidate genes in SHH signal production and transduction using RNAi-mediated inactivation or CRISPR-mediated gene ORF disruption. Consequences on production and excretion of SHH will be assessed, quantitatively and qualitatively, by measuring the ability of SHH to trigger expression of its target genes. Conversely, involvement of candidate genes in signal transmission will be determined by analysing SHH-target gene expression in inactivated receiving cells upon addition of SHH signal.

In a second approach, we will address oligogenic model by comparing molecular consequences of variant combinations to that of single events. Secreting and receiving cells will be used in a co-culture setup that allows cell-to-cell SHH signal communication. Pathogenic variants will be created through CRISPR mediated genome edition. We will take advantage of the ability of human somatic cells to reprogram into induced pluripotent stem (iPS) cells. IPS cells will be derived from peripheral blood mononuclear cells available from our biobank and will be generated at the iPSC core facility of Nantes. Once obtained iPS cells will be used to generate brain organoids by our collaborator Jürgen Knoblich (Austria) who pioneered development of cerebral organoids. Brain organoids are able to recapitulate many aspects of embryonic brain development including those related to SHH-D. Achieving these goals will allow improving diagnosis and genotype-phenotype correlation for SHH-D disorders and proposing novel genes involved in SHH signaling pathway and brain development. Notably, It will generate novel insights into the oligogenic inheritance model.