MODELOS DE REGRESIÓN DE ELECCIÓN CUALITATIVA

The XLA phenotype in family P had been found to be caused by a premature stop codon causing the protein to be terminated 24 amino acids from the end. This protein would be predicted to be approximately 70kD compared to the 77kD normal Btk. Monocytes were purified from the blood of patient CP, his obligate carrier mother and a normal control. The monocytes and the remaining mononuclear cells (predominantly B cells and T cells) were subjected to hypotonic lysis. Western blot analysis of these samples using the Btk anti serum showed the presence of the 77kD Btk protein in the monocytes and T/B cells of both the mother (MP) and the normal control and a band of the predicted size

Figure 6.3 Analysis of Btk protein in patient 276

(a)

Btk

276 D

Btk

(a) W e s te r n blot a n aly sis o f the Btk p ro tein fr om an EB V tr a n s f o rm e d B cell line from p a tie n t 2 76 an d a Daudi cell c o n tr o l , with the 7 7 k D Btk p rotein indicate d by an a r r o w on the left a nd (b) an a u to r a d io g r a p h o f an in vitro

k in ase a s s a y s h o w in g the activity o f the Btk p ro tein from patient 276 c o m p a r e d to a D a u d i c o n tr o l. Sizes o f m a r k e r s a r e g iv en in k D and are indic ate d by a r r o w s on the right.

Figure 6.4 Analysis of Btk protein from patient CP (a) D C P I MPI C l CP2 MP2 C2 Btk (b)

-Ml #

#

(a) W e s te r n b lot analysis o f h y p o to n ic lysa te s, using the Btk an tise ra. S a m p le s s h o w n a r e D a u d i cell line (D), n o n - a d h e r e n t m o n o n u c l e a r cells fro m the p e r ip h e r a l blood o f X L A p a tie n t C P ( C P I ) , his o b lig a te c a r r i e r m o th e r M P ( M P I ) a n d a n o rm al co n tro l C ( C l ) and m o n o c y t e s from the p e r ip h e r a l b lo o d o f p a tie n t C P ( C P 2 ) , his m otlier ( M P 2 ) and n o rm al c o n tr o l C (C 2). (b) Anti-Btk im m u n o p r e c i p it a te s f r o m the sa m e cell t y p e s C P I , M P I , C l , C P 2 , M P 2 a nd C 2 . (c) A u to ra d sh o w in g the ki nase activity o f the a n ti-B tk i m m u n o p r e c i p it a te s sh o w n in (b). Size m a r k e r s are sh o w n on the ri&zht in k l).

(70kD) in the monocytes of patient CP (Figure 6.4a). Additional cross-contaminating bands, which were also seen in the Daudi control, were seen in this track.

The Btk protein was immunoprecipitated from additional samples of the same types of cells from these three individuals, and subjected to a kinase assay as described in section 2.15.5. Western blot analysis of these samples showed the presence of the 77kD Btk protein in both samples from the mother (MP) and the normal control, but the absence of this protein in both samples from the affected boy (Figure 6.4b). Autoradiographic analysis of the membrane showed that the 77kD protein in the samples from the mother and the normal control had detectable in vitro kinase activity, but this activity was absent from the samples from the affected boy (Figure 6.4c). This analysis was performed twice, on separate occasions, with the same results.

The same analysis was performed on blood from patient CHD. The XLA phenotype in this patient was shown in this study to be caused by the deletion of exon 18 (Chapter 5). The results from this analysis of the Btk protein showed the complete absence of detectable protein as shown by western blot analysis of lysates, immunoprécipitation and kinase assays (results not shown). It became apparent, however, that the results of the immunoprécipitation of Btk from the monocytes of normal controls was not consistent.

6.3 Discussion

6.3.1 Involvement of BTK in other B cell immunodeficiencies

This study has involved a preliminary investigation into the potential involvement o f BTK

in disease phenotypes other than XLA. It is possible that mutations in BTK may be involved in mild forms of B cell immunodeficiencies, which may not always be clinically diagnosed as XLA. Amino acid substitutions at the less conserved residues in the protein may only have mild effects on the protein function. The case of hypogammaglobulinemia studied here did not have any alterations in the BTK gene by SSCP analysis, but another patient who was initially reported as having 5% B cells has been found to have a SSCP

band shift in the 195bp fragment of reaction A (Dr H.B.Gaspar, personal communication) indicating the presence of a mutation. The exact nucleotide change has not yet been elucidated. This fragment is the same as the one containing a mutation in Family F, who showed a milder form of XLA (Section 5.3.1).

Two patients with xid-Wkt phenotypes were investigated for mutations in BTK. No mutations were found in either of these patients by SSCP analysis. This does not completely exclude the possibility of the involvement of BTK in this phenotype as SSCP does not detect 100% of mutations (see section 5.2.1). Recently, however, a severely affected XLA patient has been described with amino acid substitutions at the same residue as the substitution found in the xid mouse (de Weers et al. 1994b). Amino acid 28 is changed to histidine in the XLA patient and cysteine in the xid mouse. Histidine and cysteine are thought of as very similar amino acids, and therefore it could be predicted that these amino acid substitutions will not have vastly different phenotypic effects. The differences in immunological phenotype may therefore be due to the different mechanisms of B cell ontogeny in the two species.

A patient previously diagnosed as having common variable immunodeficiency was found to have a genomic deletion of the BTK gene by Southern blot analysis (Vorechovsky et al. 1993), illustrating that patients diagnosed as having immunodeficiencies other than XLA may have mutations in this gene.