Gene discovery for early diagnosis of rare primary immunodeficiencies

Originality and clinical need
The majority of patients currently managed by clinical immunology have rare disorders; however within this group there are many who do not have a clear diagnosis. Some of these patients have demonstrable abnormalities of the immune system which can be life-threatening or cause significant morbidity, but which do not fit any diagnostic criteria. Even within the group of patients who do have a recognised diagnosis of primary immunodeficiency (PID), this diagnosis is frequently a clinical one without full understanding of the molecular basis of the condition. This results in difficulties selecting the correct treatment options, or advising patients about their long-term outcome.

We have used next generation sequencing technology for the diagnosis of complex patients presenting to paediatric and adult immunology clinics. In selected cases this has led to rapid diagnosis and improved clinical decision making. Currently we have several families who are being screened for novel monogenic disorders. The aim of this project is to understand the functional consequences of some of the important novel genetic mutations we have identified to date, and to explore the underlying molecular mechanisms responsible for disease in the remaining families.

Methods
Whole exome sequencing (WES) will be performed, using next generation sequencing to identify putative disease causing mutations. Pathogenic mutations in candidate genes will be assessed as appropriate, by:
a) Segregation with disease and frequencies in ethnically matched controls
b) Molecular modeling
c) Effects on splicing patterns and products in vivo
d) Expression patterns, both at the mRNA and protein level in vivo
e) Recombinant protein expression
f) Depending on the proposed function of the disease associated protein, additional methods such as immunofluorescence microscopy and flow cytometry will be used to further explore consequences of the mutation(s)



Ethical approval
The work for this project is covered by ethical approval that was granted for a study entitled “Molecular genetic investigations of autosomal recessive conditions”, which was given by the South Yorkshire Research Ethics Committee on 18th February 2011 for five years (REC ref. number 11/H1310/1). NHS Permission for Research at Leeds Teaching Hospitals NHS Trust was granted on 29th April 2011 (LTHT R&D number CG11/9764).