The m ajority o f XSCID carrier females bear an IL2RG m utation that precludes production o f stable protein, in addition to a w ildtype copy o f the gene. If m onoc\ tes
exhibit random X -chrom osom e inactivation patterns and yc chain is not required for the developm ent/differentiation and function o f this lineage, one w ould expect to observe a
m osaic pattern o f yc chain expressing and non-yc chain expressing cells (Fig 5.1). A nalysis o f the m osaicism o f expression in these lineages o f carrier fem ales may pro\ e inform ative for the process o f carrier detection. H ow ever if yc chain is important
developm entally or functionally and yc chain expressing cells have a selective growth advantage over non-expressing cells, purely yc chain expressing cells will be observed
(Fig 5.1). The applicability o f this approach has been dem onstrated in female carriers o f XLA, a disease resulting in defective B cell expansion and differentiation, caused by m utations in the gene encoding B ruton's tyrosine kinase (BTK). Female carriers exhibit apparent non-random X -inactivation patterns in B lym phocytes and random X- inactivation in non-B haem atopoietic cells such as monocytes. Clear bim odal or mosaic patterns o f BTK expressing and non-expressing m onocytes w ere seen in 85% o f the carrier fem ales studied by intracellular flow^ cytom etric analysis (Futatani et al., 1998).
In this study, four XSCID carrier females w ere analysed for cell surface yc chain expression on m onocytes by flow cytometric analysis (2.8). Their affected boys had no yc chain expression due to the nature o f the mutation (chapter 3). The aim was to
determ ine the applicability o f m onocytic yc chain expression analysis as a carrier diagnostic tool, in addition to establishing the im portance o f yc chain in m yeloid growth, differentiation and survival.
5.2 R esults
The fem ales investigated in this study are all know n to be XSCID carriers bearing
disease causing m utations that preclude production o f stable cell surface yc chain protein. Carrier M V has a strong family history o f X -linked patterns o f inheritance o f XSCID and has two XSCID affected boys, patients OV and N Y (Fig 5.2). The IL2RG m utation abolishes cell surface expression as indicated by flow cytom etric analysis o f patient N Y (Fig 3.7) and patient GY (Clark, 1996). The m other o f patient JA, MA, exhibited non-random X -inactivation in her T cell population in
inactivation
Late blastocyst stage of embroyonic development
X-inactivation
Differentiation and development X-inactivation maintained in clonal desendents
o
Non-random X-inactivation patterns in cell lineages where the absence o f y^ chain expression is detrimental to the proliferation and survival o f cells
1 0 0% of cells expressing y, chain Counts Fluorescence intensity
Random X-inactivation patterns in lineages where the lack of y^ chain expression bears no importance to cell growth and survival
50:50 ratio of expressing and non-expressing cells (bimodal distribution)
y^ chain immunostaining is indicated by the green line and immunostaining with the isotype matched control is shown in blue.
Fluorescence intensity is expressed on the abscissa and the number of events on the ordinate The dotted lines indicate the population of cells that do not express surface y^, chain
ss "O n 1 X c/3
n
n
n fS*■n>
3 3 'Z incomparison to random X-inactivation o f whole blood confirming carrier status (Lester T, Clinical Genetics laboratory, ICH, personal communication). The carrier status o f patient DC’s mother MC, and aunt was demonstrated by H in fl restriction enzyme digestion o f the 270bp PCR amplified exon 2, using primers documented in Clark et a l,
1995 (Fig 5.3). The 84 and 186bp fragments derived from wildtype DNA were easily distinguishable from the mutant 223bp fragment by 2% agarose gel electrophoresis. The mother o f patient DC carries a copy o f the mutation whereas the aunt carries two copies o f the wildtype gene. The nature o f patient D C ’s mutation prevents intracellular and cell surface expression o f yc chain protein (Figs 3.1 and 3.2).
Fig 5.2: Partial Pedigree of the patients OV and NV
s .
OV NV
Pedigree symbols are as follows: Squares-males, circles-females, filled squares- individuals affected by XSCID, filled circles-XSCID carriers, diagonal slashes- deceased.
The carrier status o f MB, who has had one affected child, patient BE, has been confirmed by analytical restriction enzyme digestion, due to the disruption o f a Alu I restriction site (Clinical Genetics, ICH, personal communication). Patient BB exhibited severely reduced levels o f yc chain expression on monocytes (Fig 3.15).
In all cases, levels o f yc chain expression by PBM NCs from carrier females were not significantly reduced from normal levels o f expression. Flow' cytometric analysis o f yc chain expression was performed on CD 14 expressing monocytes derived from PBMNCs from carriers and controls (Fig 5.4). Monocytic yc chain expression in
Fig 5.3: Carrier status determination of the mother and aunt of patient DC B Ikb ladder (bp) 5 0 6 /5 1 7 Normal Hin fl 84bp I86bp "cr Exon 2 Patient DC Hinü. 84bp 223bp
/::y:Exon 2 Mutation Exon 2
A) The partial pedigree o f the family indicating patient DC (3), the mother o f patient DC (4) and the aunt o f patient DC (5).
B) Agarose gel electrophoresis analysis o f the fragments resulting from Hin fl digestion o f exon 2 amplified from genomic DNA from these individuals. The positions o f the Ikb ladder bands are indicated on the left-hand side. The 84bp fragment was too small to be visualised in this analysis. Patient D C ’s mother bears a copy o f the mutant gene in addition to the normal gene indicated by
186bp and 223bp fragments whereas patient D C ’s aunt only bears the wild type fragment (186bp).
Lanes; 1) Ikb ladder 2) Normal control 3) Patient DC
4) M other o f patient DC 5) Aunt o f patient DC
C) Restriction map o f the PCR product encompassing exon 2 which indicates the H in fl site which generates fragments o f 186bp and 84bp seen in normal and the 223bp fragment observed in carriers and patient DC.
Fig 5.4: Yc chain expression on PB-derived monocytes from XSCID carrier females and normal controls.
XSCID Carrier icP- 10^- 10' 10» icy* 99%
i
■ A 1 % I r Yc chain lO^- expression 1 0' - 10» 10* B W - 99% iVi 10^ - 10^- 10' - 10» 1% 78% ■ ■ ^ 2 2% Normal Control 99% I» fl" 1 r.1 .-*1 * 99% 1 %88
% y 12% 10» 10' 10^ 10) i(y*io» 10' 10^ 10) 10* CD 14 expression --- ^t
Relative cell numbers 10" IQ) 10* 1C)» 1 0 ' 10" _ Yc chain expressionXSCID carrier females (a) MA, (b) MC, (c) MB (d) and MV were analysed for monocytic Yc chain expression. The PBMNC monocyte population was selected by electronic gating. Yc chain expression was detected by immunostaining with PE-conjugated anti-Yc chain monoclonal antibody, in conjunction with the monocytic lineage marker, anti-CD 14 FITC antibody. Quadrant settings distinguishing positive immunofluorescence from
background fluorescence were determined by staining with isotype-matched control antibodies. The anti-Yc chain antibody fluorescence intensity is expressed on the ordinate and the CD14 antibody fluorescence intensity is expressed on the abscissa, both on log scales. Yc chain expression by carrier M V’s monocytes is shown by the pink line and immunostaining with the isotype matched control, is shown by the green line.
PBMNCs derived from carrier females MA and MC was indistinguishable from normal levels. 8 8% o f carrier M V ’s monocytes expressed a detectable level o f yc chain in
comparison to 99% o f the normal control monocytes and 78% o f monocytes from carrier BB expressed protein compared to 8 8% in the normal control.