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Saurabh Kulkarni - Genetic causes of congenital heart diseases

Cell Biology
Supervising Faculty Member: 
Kulkarni, Saurabh
Research Focus: 

Understanding the genetic causes of congenital heart diseases

Position Description: 

Congenital heart disease (CHD) affects nearly 40,000 births per year in the United States. Despite significant improvements in perinatal, operative, and perioperative care, the morbidity, and mortality rates for infants with CHD remain unacceptably high1. Phenotypically similar patients can have dramatically different outcomes. A key reason is that current clinical care is guided by the phenotype instead of an individual's genotype. Recognizing this challenge, many clinicians are now utilizing the advances in genomic sequencing to identify pathogenic variant(s), establish a diagnosis, provide disease-specific therapy, and counsel at-risk families about recurrence risk. However, the yield of current clinical genetic testing is only about 30%. Determining pathogenicity of remaining candidate genes remains a major obstacle because most identified variants are completely novel, or overlap genes where the identified cellular function does not adequately explain the patient’s phenotype. Basic scientists can tackle this challenge by functionally analyzing these candidate genes in model organisms to support disease causation. Unfortunately, clinicians and basic biologists often work in parallel as different entities since their professional goals differ prima facie. To overcome this significant challenge, we have formed a multidisciplinary collaboration between the clinicians, bioinformaticians, and basic biologists to conduct a bench-to-bedside (B2B) project with the primary objective of improving the diagnosis and care of CHD patients at the University of Virginia Children’s Hospital (UVA Children’s) by uncovering the cellular and developmental functions of new candidate genes and further elucidate allelic heterogeneity of known CHD genes in the context of the disease phenotype. 

We have completed and approved IRB for the project and have recruited a patient with complex CHD for the study. The first step of the project to sequence the exome of the patient and their parents is complete and we have identified a candidate gene with two variants (mutations) of unknown significance present in the parents and the patient. The next step is to functional validation of these variants using the high-throughput model system of Xenopus. The goal is to provide parents with information about the probably genetic cause of disease in their offspring.

The specific objective for the student will be:

1.    Knockout and knockdown the candidate gene using CRISPR/Cas9 and morpholino oligo in Xenopus to assess if we can recapitulate the patient phenotype.
2.    Use the wildtype allele and the mutant alleles to rescue the disease phenotype to examine if the specific mutation (variant) is pathogenic.
3.    Examine the protein localization of candidate genes and assess if and how the mutations affect the protein localization.
4.    Identify the molecular cascade driven by the candidate gene and how that molecular cascade is disrupted by the mutations to lead to the disease phenotype.

Required Skills: 

Students with ambition, drive, and excellent work ethic.

Training will be provided
What will you learn: 

Examples of techniques that will be used by the student: microinjections, genetic manipulation using CRISPR/Cas9, confocal/super-resolution microscopy, cloning, protein localization using immunofluorescence, protein quantification using western blots, and functional analysis of mutants using pull-down assays.