Project Title: „Functional and genetic research of rare unidentitied neuromuscular disorders”

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project No.: 1.1.1.1/18/A/097

Period: 1 April 2019 – 31 March 2022

Project costs: 648 648 EUR

Principle Investigator: Dr. biol. Inna Iņaškina

Cooperation partner: SIA Latvia MGI Tech

Project summary:

The general aim of the project is to improve public health through the expansion of our knowledge about rare neuromuscular diseases and diagnostics of patients with such disorders. The specific aim of the project focuses on two topics: 1) to better understand and characterize the recently identified mutant MYBPC1 protein, its interactions with other cellular partners, and the molecular mechanisms underlying newly described disease phenotype; 2) using the newest molecular biology methods, namely whole genome sequencing, to identify novel causative genetic elements (genes, inter-gene regions or chromosomal rearrangements) for rare and/or unidentified neuromuscular disorders.

To reach the specific aim of the project, we have tailored the following activities: development of knockin mouse model for the mutation in human MYBPC1 gene and its in-depth study; transcriptome analysis of patients’ tissue samples and tissue samples obtained from the mouse models; recruitment of patients suffering from rare and/ or unidentified neuromuscular diseases; whole genome sequencing analysis of aforementioned patients with a goal to identify novel causative genetic elements.

Research will join advances and technologies from several scientific fields: medicine, biomedicine, biotechnology and bioinformatics. Public access to research results will be provided in the form of animal functional model data, publications and patients’ whole genome sequencing data available for further studies (in an anonymized form).

Information published 01.04.2019.

Progress of the project:

1 April 2019 – 30 June 2019

In the frame of the ERDF 097 project, the analysis of the mutant MYBPC1 protein mouse model was continued in cooperation with the Dr A.Controgianni group (University of Maryland School of Medicine), the obtained data were presented at the European Human Genetics Conference in June in Gothenburg, Sweden. In parallel, a procurement procedure is being prepared for the production of a new mouse model.

As part of the project, the involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was started. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed.

Information published 28.06.2019.

Progress of the project:

1 July 2019 – 30 September 2019

In the frame of the ERDF 097 project, the analysis of the mutant MYBPC1 protein mouse model was continued. The RNA profile (transcriptome) analyses of different functionality muscle tissues, comparing mutant mice with the control group, were started. The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed.

Information published 30.09.2019.

Progress of the project:

1 October 2019 – 31 December 2019

In the frame of the ERDF 097 project, the analysis of the mutant MYBPC1 protein mouse model was continued.  The data obtained in cooperation with the Dr A.Controgianni group (University of Maryland School of Medicine)  were presented at the American Human Genetics Conference in October in Houston, Texas. The RNA profile (transcriptome) analyses of different functionality muscle tissues, comparing mutant mice with the control group, were continued. The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives.  The data analysis algorithm is currently being developed.

Information published 30.12.2019.

Project progress

 1 January 2020 – 31 March 2020

In the frame of the ERDF 097 project, the analysis of the mutant MYBPC1 protein mouse model was continued. The transcriptome analysis of muscle tissues from different muscle types, comparing mutant mice with the control group, has been completed. Based on that several genes that exhibited statistically significant difference in the expression level have been selected for further validation using ddPCR and Q-PCR approaches.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives.  The data analysis algorithm is currently being developed.

Information published 31.03.2020.

Progress of the project:

1 April 2020 – 30 June 2020

In the framework of the ERDF 097 project, the analysis of the mutant MYBPC1 protein mouse model is being continued. Due to the emergency caused by the COVID-19 epidemic, work in the laboratory was suspended in March and resumed in June. The results of transcriptome analysis of muscle tissues from different muscle types are currently being validated, using alternative molecular biology methods (ddPCR and Q-PCR)

The involvement of patients, suffering from inherited neuromuscular diseases, and their relatives in the Genome Database of Latvian Population was continued. Due to the emergency caused by the COVID-19 epidemic, the patients’ involvement was suspended in March and resumed in June.

Joint meetings between project executors and participating clinical specialists (in person and on-line) are being held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing). The analysis of previously obtained data is currently being performed.

Information published 30.06.2020.

Progress of the project:

1 July 2020 – 30 September 2020

Within the framework of the ERDF 097 project, the analysis of the mutant MYBPC1 protein mouse model was continued. The findings for several genes that have been selected based on transcriptome analysis of muscle tissues from different muscle types (Ky, Kcnc1, Mylk4, Myl6) are currently being validated, using Droplet Digital PCR approach.

Furthermore, a new knock-in mouse model for the second MYBPC1 mutation (p.Y247H), exhibiting the same disease phenotype, has been created and is currently undergoing final testing for specific pathogens. Upon arrival we are planning to establish transgenic mice colony here in Latvia and to analyse the transgenic animals comprehensively.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives. The genetic diagnosis was established for four patients and several potentially pathogenic genetic variants have been found in others. Further analysis is necessary. The data analysis algorithm is still being developed and improved.

Information published 30.09.2020.

Progress of the project:

1 October 2020 – 31 December 2020

Within the framework of the ERDF 097 project, a new knock-in mouse model for the MYBPC1 mutation (p.Y247H), exhibiting the same disease phenotype, has been created and underwent final testing for specific pathogens. The transgenic animals have been delivered to Latvia in the beginning of November. Upon arrival we start to establish transgenic mice colony here in Latvia – the first transgenic pups were already delivered.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives. The genetic diagnosis was established for four patients and several potentially pathogenic genetic variants have been found in others. Further analysis is necessary. The developed data analysis algorithm is still being improved.

Information published 30.12.2020.

Progress of the project:

1 January 2021 – 31 March 2021

Within the framework of the ERDF 097 project, we have stablished transgenic mice colony of a new knock-in mouse model for the MYBPC1 mutation (p.Y247H), at the moment it consists of more than 50 animals.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives. Several potentially pathogenic genetic variants have been found in some patients. Further analysis is necessary. The developed data analysis algorithm is still being improved. In parallel, available row data from patients’ previous genetic tests, for example, whole exome or gene panel sequencing, are re-analysed.

Information published 31.03.2021.

Progress of the project:

1 April 2021 – 30 June 2021

Within the framework of the ERDF 097 project, we have stablished transgenic mice colony of a new knock-in mouse model for the MYBPC1 mutation (p.Y247H), at the moment it consists of more than 80 animals.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives. Several potentially pathogenic genetic variants have been found in some patients. Further analysis is necessary. The genetic diagnosis was established for several patients.

The obtained data on rare pathogenic genetic variants and connected diagnoses was presented in frame of 79th International Scientific Conference of the  University of Latvia as oral presentation: B. Lāce et al. “Calpain-3-related limb-girdle muscular dystrophy: complex genetics for a rare disease”.

Information published 30.06.2021.

Progress of the project:

1 July 2021 – 30 September 2021

Within the framework of the ERDF 097 project, we have maintained transgenic mice colony of a new knock-in mouse model for the MYBPC1 mutation (p.Y247H), as well performed several functional tests for animals at different age (2, 4 and 8 weeks).

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Joint meetings between project executors and participating clinical specialists are held regularly, where cases of individual patients are discussed and decisions are made whether to move them to the next phase of the project (whole genome sequencing).  The whole genome sequencing analysis was performed for selected patients and their relatives. Several potentially pathogenic genetic variants have been found in some patients. Further analysis is necessary. The genetic diagnosis was established for several patients.

Information published 30.09.2021.

Progress of the project:

1 October 2021 – 31 December 2021

During the reference period, we continued transgenic mice colony maintenance of a new knock-in mouse model for the MYBPC1 mutation (p.Y247H, total number of animals reached 110 pcs), as well as worked on new analysis of these transgenic animals. Muscle strength tests (righting reflex, inverted hang) were performed, as well as functional (open-field, grip force, rotating rod) tests. The dynamics of weight gain and determination of tremor in new-born babies were also performed. As well as muscle samples collected for a variety of studies, for analysis of transcriptome RNAseq, electron microscopy and immunofluorescence analyses.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population was continued. Samples of selected patients and their relatives were transferred to our partner MGI Latvia for whole genome sequencing. Analysis, annotating and interpretation of the data and validation of obtained variants were performed. Several potentially pathogenic genetic variants have been found in some patients. Further analysis is necessary. The genetic diagnosis was established for several patients.

Information published 30.12.2021.

Progress of the project:

1 January 2022 – 31 March 2022

During the reference period, we continued transgenic mice colony maintenance of a our knock-in mouse model for the MYBPC1 mutation p.Y247H (total number of animals reached 240 pcs), as well as proceeded with analysis of these transgenic animals.  Muscle samples were collected for a variety of studies – electron microscopy, immunofluorescence and other analyses. A transcriptome analysis using RNAseq was conducted on muscle tissue samples from Y247H and wt animals.

The involvement of patients suffering from inherited neuromuscular diseases and their relatives in the Genome Database of Latvian Population (LGDB) was continued. The total number of patients suffering from inherited neuromuscular diseases and their relatives reached 99. In addition, around 400 single individuals suffering from different diseases, including very rear (but without proved inheritance factor) were also recruited into LGDB.  Samples of selected patients and their relatives were transferred to our partner MGI Latvia for whole genome sequencing. Analysis, annotating and interpretation of the data and validation of obtained variants were performed. Several potentially pathogenic genetic variants have been found in some patients. Further analysis is necessary. The genetic diagnosis was established for several patients.

A retrospective study “Overview of neuromuscular disorder molecular diagnostic experience for the population of Latvia” summarized more than a decade of clinical evaluation and genetic testing of NMD patients in Latvia which also included some findings that resulted from our project. The collected results hypothesized that congenital myotonia caused by the CLCN1 gene variant c.2680C>T p.(Arg894Ter) is the most frequent NMD in Latvia (Lace et al., accepted in journal Neurology. Genetics).

Information published 31.03.2022.