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Results WP2: The Genetic Etiology of PADs

Linkage analysis in antibody-deficient families

Linkage analysis is a non-hypotesis-driven pure positional cloning approach. By linkage analysis, the consortium was successful in identifying a novel genetic cause for CVID in four autosomal recessive families from Belgium, Israel and Iran. The causative gene was previously not known to be important for the immune system and most likely affects autophagy of lymphocytes. What is specially interesting for the pathophysiological understanding is the fact that the phenotype in these patients include autoimmunity. This paper is currently under revision. In the other CVID families we located the genetic defect to certain chromosomal regions and positional candidate genes were sequenced. The autosomal-dominant CVID family FR1 is linked to chromosomes 5, 6 and 7. The autosomal-dominant CVID family NL1 is linked to chromosome 4. The autosomal-dominant CVID family FR15 is linked to chromosome 3. The autosomal-recessive CVID family US2 is linked to chromosomes 1, 5 and 20. The autosomal-recessive CVID families Port1 and cv32 have multiple regions of homozygosity as they are quite small. Finally, the autosomal-dominant CVID family IT4 is linked to chromosome 4 and achieved a LOD score of 3.9. However, as this purely positional approach followed by Sanger sequencing of possible candidates in the linkage regions was not frutful in these families for the last few months. Hence we are now employing whole exome sequencing to identify the genetic defect. This will be carried out in part by the EU project EURO GENEscan where UCL and KI are partners.

Genome-wide association studies

Tissue typing on Iranian and Swedish CVID patients/families reveals that the previously suggested HLA association in CVID/IgAD multicase families is largely restricted to those carrying the HLA B8, DR3, DQ2 haplotype and that the pattern in "sporadic" CVID patients is largely random. The addition of 100 more cases confirmed the initial findings. Analysis of the contribution of genes within the HLA region in 100 multicase IgAD families is ongoing at KI (analysis of degree of HLA sharing among affected/non-affected siblings of the proband). This material is also currently being extended, including additional multicase families. A preliminary analysis, using the formula of Risch, has suggested that genes within the MHC region contribute approximately 40% of the disease risk in IgAD.

The Candidate Gene Approach

In an exceptional effort, fourteen functional candidate genes for CVID were studied by a single team in Brescia, Italy; some of which may represent disease-causing genes, others may be modifier genes for CVID.

The Genetics of Hyper-IgM Syndromes

Whole exome sequencing in five patients from different families with hyper IgM syndromes of unknown cause was performed. In two siblings we found a mutation in the CD40L gene, although CD40L-deficiency seemed previously excluded by normal CD40L expression. This observation highlights the fact that protein detection alone does not exclude a mutation. In other two families, two genes were found mutated on both alleles. These genes encode proteins involved in both chromatin modification and DNA repair. These mutations are found "likely damaging" in Polyphen database and are not described as polymorphisms in databases. No murine model is available. Mutations in both genes are now looked for in a large cohort of HIGM patients (80 patients). To validate the role of these molecules in CSR, shRNA lentiviral transduction of CSR-induced CH12 cell line is now on-going. In another non-consanguineous family, with two affected children, we found in both patients a few genes carrying bi-allelic mutations, that are now under investigation. Five other informative families have been studied by exomic sequencing and results are pending. In a consanguineous and informative family, in which a homozygous genetic region has been already determined, a gene was found mutated as homozygous in the two affected children. However, a normal sibling and the healthy mother present the same genotype, excluding the role of this mutation in the pathogeny of the disease. Consequently, all the exons present in this region, not completely or not perfectly covered by the exome, were sequenced, with no detected abnormalities. Expression of the cDNA level of all the genes comprised in that region will be assessed as soon as material is available (EBV B cell line) in order to detect splice defects (due to intronic mutations) or defective expressions (mutations in promotor).