Screening of functional and positional candidate genes in families with common variable immunodeficiency

Seventeen probands of CVID families were analyzed. Seven of these families were selected due to being linkage-positive at the TNFRSF17 locus on chromosome 16p (Table 1). In addition, we expanded our mutation screening in BCMA/TNFRSF17 to a larger cohort of 50 sporadic CVID cases. One previously described heterozygous single nucleotide polymorphism (SNP) was found in a proband of family cv22 in exon 2: Ser81Asp [dbSNP: rs373496]. In exon 3 two synonymous SNPs [dbSNP: rs2017662 and rs2071336] were found in homozygous state in five individuals (family fr24 and four sporadic cases) and in heterozygous state in probands of families cv73 and fr21. RT-PCR was performed but evidenced no alternative splicing products (data not shown). A novel variant was identified in exon 3: K179Q, which was present in one individual. The K179Q variant was present in the heterozygous state both in affected and healthy members of the family, which renders unlikely a contribution of this mutation to the pathogenesis of the hypogammaglobulinemia. Flow cytometric analysis confirmed normal expression of the protein (data not shown).

APRIL/TNFSF13 was analyzed in the probands from 12 CVID families. The families cv73, cv97 and cv137 were linkage positive at the TNFSF13 locus (Table 1). Two previously described non-synonymous SNPs [dbSNP: rs11552708 and rs3803800], located in exon 1 and exon 2 respectively, were found in the studied cohort (Table 3).

The IL10 gene was sequenced in 23 individuals. Of those, 13 represented autosomal recessive CVID families, five families were selected for being linkage-positive, and five were sporadic CVID cases showing a clinical phenotype compatible with inflammatory bowel disease (Table 1). One previously described heterozygous SNP [rs3024496] located in the 3'UTR of the IL10 gene was found in frequencies comparable to that in healthy controls (Table 3).

IL10 receptor α, encoded by IL10RA was analyzed in 22 individuals, of whom 12 belonged to autosomal recessive families, five were from linkage-positive families at the IL10RA locus, and five were sporadic CVID cases (Table 1). The known synonymous SNP [dbSNP: rs2256111] in exon 4 and the SNP [dbSNP: rs17121493] in exon 5 (I224V) were observed in frequencies comparable to those which are reported for normal controls in public databases (Table 3).

The IL10RB gene was analyzed in 14 CVID patients, 10 from CVID families (six of them linkage-positive at the IL10RB locus) and four sporadic CVID cases (Table 1). We found no genetic alterations in the 7 exons of IL10RB.

The 5 exons of IL21 were amplified and sequenced in 25 individuals (19 autosomal recessive CVID (AR-CVID) families and 6 autosomal dominant CVID families (AD CVID) that are linkage-positive at the IL21 locus). In exon 3 of the IL21 gene the synonymous change [dbSNP: rs4833837] was observed in a frequency comparable to that reported in public databases (Table 3).

The IL21R gene was analyzed in 24 individuals (19 AR CVID families and 5 AD CVID families linkage-positive at the IL21R locus). In exon 1 of IL21R the SNP [dbSNP: rs961914] was observed in homozygous state in the proband from family fr18 and in heterozygous state in cv32, fr25, fr28 and fr29. In exon 5 of the IL21R gene we found a previously undescribed heterozygous SNP resulting in the replacement of a conserved threonine by methionine at position 46 in the proband from family cv4 (Figure 3). In addition, a new heterozygous change in exon 8 resulting in R275Q was found in the family cv72. Both heterozygous substitutions showed perfect segregation with the disease phenotype in these families (Figure 3). However, we subsequently screened a cohort of 100 healthy individuals with normal immunoglobulin levels and found respectively 2 (T46M) and 5 (R275Q) heterozygous individuals among them, suggesting that these two coding sequence changes may not be disease-associated.

The three exons of CCL18 including the 5'and 3'untranslated region were amplified and analyzed in 19 probands including six from CVID families linkage-positive at the CCL18 locus on chromosome 17 (Table 1). No genetic alterations were found.

For several of the genes we mention, such as TACI/TNFRSF13B, the HUGO-approved gene acronym (TNFRSF13B) has no resemblance to the protein acronym (TACI), which is much more familiar to immunologists. In those cases, we use either the protein name alone or the compound notation protein/gene, when referring to either the gene or the protein it encodes.

Twenty families with at least one case of CVID were selected from a collection of 101 multiplex CVID/IgA deficient families [5, 9, 10, 34], based in part on reanalysis of genotype data previously generated for genetic linkage studies. This method had successfully identified family A as consistent with linkage to the TACI/TNFRSF13B locus [15]. In addition, 19 autosomal recessive CVID families were collected at the Department of Rheumatology and Clinical Immunology in Freiburg, Germany, from 2001 to 2005. The families were considered as having possible recessive inheritance when two or more siblings in one generation were affected by CVID and their parents and children had normal immunoglobulin levels. Alternatively, ten families were considered as recessive because of a known consanguinity in these families. Six families originated from Turkey, five from Germany, four from Italy, two from the UK and one from Sweden. For the analysis of TNFRSF17, encoding BCMA, fifty additional individuals with sporadic CVID followed in the Department of Pediatrics, University of Brescia were included in this study. Informed written consent was obtained from each patient or parent guardians prior to participation under the internal ethics review board-approved clinical study protocol (ZERM, University Hospital Freiburg (#239/99).

Genetic linkage analysis was carried out by computing LOD scores using a dominant model and imperfect penetrance using FASTLINK [35‐37], as previously described [11]. We also tried a recessive model in which the penetrance for heterozygotes is assumed to be the same (0.05) as for individuals with no disease-associated alleles. For each candidate gene, we tried to find close flanking markers from the marker set chosen for the previous studies [9, 10]. The relevant markers near each gene are shown in Table 1. We tried to find families with positive scores at all flanking markers, but sometimes had to settle for families with positive scores at one or some flanking marker(s) and 0 scores (either due to no genotypes or an uninformative marker) at some flanking marker(s). With the exceptions of cv32 and cv74, all the families are small and have maximum achievable scores under 1.0. Thus, the positive scores should be taken only as evidence that these families are consistent with linkage and preferred over families with negative LOD scores, but we make no claims of statistically significant linkage. Based on these criteria, the families which were consistent with linkage for each individual candidate gene were selected out of the previously described cohort [9, 10] (Table 1, Figure 1).

Candidate genes were evaluated by sequencing the coding regions of the genes on genomic DNA including 20 bp of the flanking intronic or untranslated regions. All primers were sought with the aid of the Primer Select software (PE Applied Biosystems, Foster City, CA, USA); sequences are summarized in Table 2. Genomic DNA was amplified by PCR and subsequently sequenced with the amplification primers. After gel electrophoresis on an ABI Prism™ 377 DNA Sequencer, the data was analyzed by the DNA Sequencing Analysis software, version 3.4 (PE Applied Biosystems) and Sequencer™, version 3.4.1 (Gene Codes Corporation, Ann Arbor, MI, USA).