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Standardised imaging pipeline for phenotyping mouse laterality defects and associated heart malformations, at multiple scales and multiple stages.

ABSTRACT: Laterality defects are developmental disorders resulting from aberrant left/right patterning. In the most severe cases, such as in heterotaxy, they are associated with complex malformations of the heart. Advances in understanding the underlying physiopathological mechanisms have been hindered by the lack of a standardised and exhaustive procedure in mouse models for phenotyping left/right asymmetries of all visceral organs. Here, we have developed a multimodality imaging pipeline, which combines non-invasive micro-ultrasound imaging, micro-computed tomography (micro-CT) and high-resolution episcopic microscopy (HREM) to acquire 3D images at multiple stages of development and at multiple scales. On the basis of the position in the uterine horns, we track in a single individual, the progression of organ asymmetry, the situs of all visceral organs in the thoracic or abdominal environment, and the fine anatomical left/right asymmetries of cardiac segments. We provide reference anatomical images and organ reconstructions in the mouse, and discuss differences with humans. This standardised pipeline, which we validated in a mouse model of heterotaxy, offers a fast and easy-to-implement framework. The extensive 3D phenotyping of organ asymmetry in the mouse uses the clinical nomenclature for direct comparison with patient phenotypes. It is compatible with automated and quantitative image analyses, which is essential to compare mutant phenotypes with incomplete penetrance and to gain mechanistic insight into laterality defects.

SUBMITTER: Desgrange A

PROVIDER: S-EPMC6679386 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Standardised imaging pipeline for phenotyping mouse laterality defects and associated heart malformations, at multiple scales and multiple stages.

Desgrange Audrey A Lokmer Johanna J Marchiol Carmen C Houyel Lucile L Houyel Lucile L Meilhac Sigolène M SM

Disease models & mechanisms 20190709 7

Laterality defects are developmental disorders resulting from aberrant left/right patterning. In the most severe cases, such as in heterotaxy, they are associated with complex malformations of the heart. Advances in understanding the underlying physiopathological mechanisms have been hindered by the lack of a standardised and exhaustive procedure in mouse models for phenotyping left/right asymmetries of all visceral organs. Here, we have developed a multimodality imaging pipeline, which combines ...[more]

PMID: 31208960

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Project description:Background and aimsMutations in KCNH2 cause long or short QT syndromes (LQTS or SQTS) predisposing to life-threatening arrhythmias. Over 1000 hERG variants have been described by clinicians, but most remain to be characterised. The objective is to standardise and accelerate the phenotyping process to contribute to clinician diagnosis and patient counselling. In silico evaluation was also included to characterise the structural impact of the variants.MethodsWe selected 11 variants from known LQTS patients and two variants for which diagnosis was problematic. Using the Gibson assembly strategy, we efficiently introduced mutations in hERG cDNA despite GC-rich sequences. A pH-sensitive fluorescent tag was fused to hERG for efficient evaluation of channel trafficking. An optimised 35-s patch-clamp protocol was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses.ResultsIn the present work, we observed a good correlation between cell surface expression, assessed by the pH-sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophysical parameters, that can all be recapitulated in a single parameter defined herein as the repolarisation power. The impacts of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarisation power and structural impact) define three pathogenicity indexes that may help clinical diagnosis.ConclusionsFast-track characterisation of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. It also helped the diagnosis of two new variants. This information is meant to fill a patient database, as a basis for personalised medicine. The next steps will be to further accelerate the process using an automated patch-clamp system.

Project description:As a rare disease with genetic pathogenesis, observational study about familial CHD recurrence risk on CHD patients with laterality defects is lacking. This study aimed to investigate familial recurrence among families of patients with CHD and laterality defects, and compare them with CHD patients without laterality defects. A total of 184 patients with CHD and laterality defects treated in Cardiovascular Center, Children's Hospital of Fudan University were observed from 2008 to 2019. A detailed family history was documented by trained staff using questionnaires, and information about the subtypes of CHD and laterality defects was also collected. In addition, positive family history information, including all three degrees relatives and all affected family members, was reconfirmed by trained medical staff through face-to-face interviews, telephone interviews, and letter return visits. Of the 184 included patients, 30 had at least one family member (from among three linear generations and distant relatives) with CHD. The familial recurrence rate of CHD in our cohort was 16.3% (30/184), which was higher than the 3.3% (67/2024) of patients with CHD without laterality defects. This result shows that the recurrence rate among the first-, second-, and third-degree relatives was 11.7% (11/94), 1.5% (3/204), and 3.1% (6/91) and that the recurrence rate among siblings (21.4%, 9/42) was higher than that among parents (3.8%, 2/52). The familial recurrence risk of CHD among patients with CHD and laterality defects is high, which is consistent with the previous study that reported a high familial recurrence of heterotaxy of 10%. First-degree relatives have a higher recurrence rate than second- and third-degree relatives, especially siblings. These findings have important significance for prenatal screening, intervention, and genetic counseling in the Chinese population, but may not be generalizable to other populations that may have different rates of familial and sporadic cases.

Dataset Information

Standardised imaging pipeline for phenotyping mouse laterality defects and associated heart malformations, at multiple scales and multiple stages.

Publications

Standardised imaging pipeline for phenotyping mouse laterality defects and associated heart malformations, at multiple scales and multiple stages.

Similar Datasets

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