Variance detection.

Due to the nature o f the techniques involved, the variance detection strategy was not applied in a serial manner; that is, all amplicons were not all generated and screened from a given sample before proceeding to the next sample. Rather a parallel processing approach was employed whereby amplicons derived from a single region o f H E SX l were generated from a number o f samples and screened as a batch. Similarly, due to the nature o f patient

recruitment and the WAVE system only becoming available during the course o f study and the ongoing investigation into H E SXl genomic arrangement, the two variance detection techniques were not applied serially. Rather SSCP detection was used on exons 1-4 from all samples recruited until the WAVE system became available. Then dHPLC-heteroduplex detection was used on exons 1-4 from samples previously screened by SSCP detection and those recruited in the meantime. When the appropriate genomic sequence became available, dHPLC-heteroduplex detection was applied to putative exons alt A and alt B from all samples

recruited at that time, followed by dHPLC-heteroduplex detection being applied to the 5’LIM l homology region. SSCP detection was then performed on exons 1-4 o f those samples recruited since the WAVE system became available. Finally, all amplicons outstanding were screened. Exons alt A, 1, 2, 3, and 4 were screened using both dHPLC- heteroduplex detection and SSCP detection. Exon alt lb and the 5 ’ LIM l homology region were screened by dHPLC-heteroduplex detection only. Finally, a small number o f amplicons could not be amplified and thus a number o f patients were not completely screened (Table 2.4.2a).

Stages Regions Method Samples

1 1-4 SSCP Samples recruited until WAVE became available.

2 1-4 WAVE Samples screened by SSCP in stage 1.

3 1-4 WAVE Samples recruited since beginning of stage 2. 4 1-4 SSCP Samples recruited since beginning o f stage 2. 5 alt A - alt B WAVE All samples recruited at time point. 6 5 ’ LIM l

homology region

WAVE Subset o f all samples recruited at time point. 7 alt A, 1, 2,3,4, SSCP All samples recruited since

beginning o f stage 5.

8 All Both Any samples outstanding

(Table 2.4.2a) S tages o f H E S X l m utation screening.

In total 5546 DNA amplifications, consisting o f 2673 SSCP detection reactions and 2873 dHPLC-heteroduplex detection reactions, were performed from sporadic cases and screened for mutations within H ESXL This figure excludes samples from which poor results were obtained due to either poor amplification, or poor variance detection, and thus amplification and screening were repeated. Based upon these results o f this variance detection protocol,

188 amplicons were regenerated and directly sequenced for changes W iihm H E SXl. This identified 14 sequence variants. SSCP detection also produced 4 false negative results as SSCP detection failed to detect 4 sequence variants that were later identified using dHPLC- heteroduplex detection. No false negative results were identified within the dHPLC- heteroduplex negative samples (Table 2.4.2b).

Region SSCP SSCP false positivel SSCP false negative* SSCP positive dHPLC dHPLC heteroduplex false positive dHPLC heteroduplex false negative* dHPLC heteroduplex positive Total screened amplifications alt A 523 13 0 0 554 13 0 1 1077

alt B n/a n/a n/a n/a 554 0 0 0 554

Liml n/a n/a n/a n/a 183 15 0 4 183

Exon 1 536 16 0 0 537 33 0 0 1073

Exon 2 541 10 2 0 530 11 0 2Î 1071

Exon 3 537 20 1 3 n/a n/a n/a n/a 537

Exon 4 536 28 1 2 n/a n/a n/a n/a 536

Exon 3/4 n/a n/a n/a n/a 515 25 0 2 515

Total 2673 87 4 5 2873 97 0 9 5546

(Table 2.4.2b) Breakdown o f mutation screening results. *known fa ls e negatives, f excludes duplex artefacts, / excludes 4489.

2.4.2.1 Putative exon alt A

A total of 554 samples were screened by dHPLC-heteroduplex detection, while SSCP detection was carried out on a total of 523 samples. dHPLC-heteroduplex detection identified 13 positive peak-shifts, 1 of which was due to sequence variance, while SSCP detection identified 13 positive band-shifts none of which were due to sequence variance. Representative putative exon alt A SSCP detection results are shown in Figure 2.4.2.1a while representative putative exon alt A dHPLC detection results are shown in figures 2.4.2.1b, and 2.4.2.1c. A result later shown to correspond to a sequence change is shown in figure

2.4.2.Id.

S S % £ fe

s S

s a v i s a » 0 »

Figure 2.4.2. la . A H ESXl exon A SSCP detection g e l show ing a sam ple with a S C C change (arrow), this is an exam ple o f a fa lse p o sitiv e result. Wild type is f a r right.

i . e R e t e n t i o n T u s e (m m )

(Figure 2.4.2. Ib) An overla y o f exon A dH PL C resu lts sh ow ing three sam ples 4883 (blue), 4884 (green), an d 4885 (red), with w ild-type (black).

3 , 2

3 .O R e t e n t i o n Tim e (m in )

(Figure 2.4.2. Ic) An overla y o f exon A dH PL C resu lts sh ow in g fo u r sam ples 4 5 1 7 (blue), 45 1 8 (green), 4519 (red), an d 4521 (black). N ote that the p e a k height o f 4518 (green) has been e q u a lised to that o f the other 3

sam ples resu ltin g in a lack o f v ertica l scale. 4 5 1 8 (green) is an exam ple o f a fa ls e p o sitiv e result.

(Figure 2.4.2. Id) An o verla y o f exon A dH PL C resu lts sh ow in g fo u r sam ples 3881 (blue), 3882 (green), 3883 (red), a n d 3 89 (black). N ote that the p e a k height o f 3883 (red) has been equ alised to that o f the other 3 sam ples

resu ltin g in a lack o f v ertica l scale. 3883 (red) resu lts fro m the sequence variant a lt -4 t> g

2A.2.2 Putative exon alt B

A total o f 554 samples were sereened by dHPLC-heteroduplex deteetion. Due to time constraints and in the knowledge that dHPLC-heteroduplex deteetion had produced no false positive results SSCP deteetion was not carried out. Representative alternative exon B dHPLC results are shown in figures 2.4.2.2a and 2.4.2.2b. dHPLC detection identified no changes suggestive o f sequence variance.

0 . 5

(Figure 2 .4 .2 .2 a) An o verlay o f exon B dH PL C resu lts f o r tw o sam ples 4857 (blue), a n d 48 6 7 (green), alongside w ild -typ e (red).

R e c « n c i . o & T X æ ( m m )

(Figure 2.4.2.2b) An overlay o f exon B dH PL C resu lts f o r fo u r sam ples 4869 (blue), 4800 (green), 4814 (red), and 4802 (black).

2.4.2 3 5’ L IM l homology region.

A total o f 183 samples were sereened by dHPLC-heteroduplex. Due to time constraints and in the knowledge that dHPLC-heteroduplex detection had produced no false negative results SSCP detection was not carried out dHPLC detection identified 15 changes suggestive o f sequence variance, 4 o f which were due to sequence variance. Representative 5’ LIMl domain dHPLC detection results are shown in figures 2.4.2.3a, and 2.4.2.3b. A result later shown to correspond to sequence change is shown in figure 2.4.2.3c.

*.s 1.0

0- 3

(Figure 2 .4.2.3a) An o verlay o f 5 ’ L IM l hom ology region dH PL C resu lts a t 6 I° C f o r fo u r sam ples 4751 (blue), 4754 (green), 4718 (red), an d 4714 (black).

(Figure 2.4.2.3b) An o verlay o f 5 ’ L lM l hom ology region dH P L C resu lts a t 59°C f o r fo u r sam ples 5 0 5 7 (blue), 5071 (green), 5072 (red), a n d 4842 (black).

4 9 4 . ft S.O ft ft I.O

1.0

(Figure 2.4.2.3c) An o verla y o f 5 ’ L lM l hom ology region dH PLC resu lts at 59°C f o r fo u r sam ples 4988 (blue), 4989 (green), 4992 (red), an d 5 001 (black). 4 9 8 8 (blue) resu lts fro m the sequence variant -2 7 7 t> g

2.4.2 4 Exon 1

A total o f 537 samples were screened by dHPLC-heteroduplex detection, while SSCP detection was carried out on a total 536 samples. dHPLC-heteroduplex detection identified 33 positive peak-shifts, none o f which was due to sequence variance, while SSCP detection identified 16 positive band-shifts, none o f which was due to sequence variance.

Representative exon 1 SSCP detection results are shown in figures 2.4.2.4a, while representative exon 1 dHPLC detection results are shown in figures 2.4.2.4b and 2.4.2.4c.

I

t

(Figure 2.4.2.4a) A H E SX l exon I SSC P detection g e l sh ow in g a sam ple with a SSC change (arrow ) a n d sam ples with duplex changes (brackets). W ild type is f a r right.

(Figure 2.4.2.4b) An o verlay o f exon 1 dH PL C resu lts sh ow in g fo u r sam ples 4015 (blue), 4 3 4 7 (green), 4578 (red), an d 3 35 (black).

R e c e n c i o n TUae

(Figure 2.4.2.4c) An overla y o f exon I dH PL C resu lts sh ow in g fo u r sam ples 4644 (blue), 46 3 9 (green), 4640 (red) an d 46 4 7 (black). 4640 (red) is an exam ple o f a fa ls e p o sitiv e result.

2.4.2.5 Exon 2

A total o f 530 samples were screened by dHPLC-heteroduplex deteetion, while SSCP deteetion was carried out on a total 541 samples. dHPLC-heteroduplex detection identified

11 positive peak-shifts, 2 o f which were due to sequence variance, whilst SSCP detection identified 10 positive band-shifts, none o f which was due to sequence variance. SSCP deteetion also failed to detect the 2 samples containing sequence variance that were later

identified by dHPLC-heteroduplex detection. Representative exon 2 SSCP detection results are shown in figure 2.4.2.5a, while representative exon 2 dHPLC detection results are shown in figures 2.4.2.5b and 2.4.2.5c. Results later shown to correspond to a sequence change are shown in figures 2.4.2.5i (183t>c), 2.4.2.5j (219c>t), and 2.4.2.5k (Y90H).

(Figure 2 .4.2.5a) A H E SX l exon 2 SS C P detection gel. W ild type is f a r left.

(Figure 2.4.2.5h) An ov erla y o f exon 2 dH PL C resu lts sh ow in g three sam ples 4900 (blue), 4870 (red), an d 4871 (black), with w ild-type (green).

20

1 0 -

12 13 14

3 7 8 9 10 11

0 1 2 4 5 6

Retention Time (min)

(Figure 2.4.2.5c) An overlay o f exon 2 dH PL C resu lts sh ow in g fo u r sam ples 4 8 2 0 (blue), 5103 (green), 5120 (red), a n d 5 134 (black). 5134 (black) is an exam ple o f a fa ls e p o sitiv e result; note that the p rim e r p e a k

an d the m ain p e a k are reta in ed longer than the other three sam ples an d that the w ash p e a k is absent.

R e t.c n c i.o n Time (m in)

(Figure 2.4.2.5d) An overlay o f exon 2 dH PL C results sh ow in g fo u r sam ples 4585 (blue), 4410 (green), 4614 (red), a n d 4624 (black). 4614 (red) resu lts fro m the sequence variant 183t>c. N ote the fa ilu re o f 4624

(black).

R e t e n t i o n T i n e m i n i

(Figure 2.4.2.5e) An overlay o f exon 2 dH PL C resu lts sh ow in g fo u r sam ples 4203 (blue), 3711 (green), 3936 (red), an d 4212 (black). 4212 (black) resu lts fro m the sequence variant 219c> t.

1

-rrrf

(Figure 2.4.2.5f) An o verla y o f exon 2 dH PL C resu lts sh ow in g fo u r sam ples 4954 (blue), 4214 (green), 4352 (red), an d 4489 (black). 4489 (black) resu lts fr o m the sequence variant Y90H.

2.4.2.6 Exon 3 and 4

A total o f 537 samples were screened by SSCP detection for variance within exon 3, while 536 samples were screened by SSCP detection for variance within exon 4. The small size o f intron 3 allowed both exon 3 and exon 4 to be screened together by HPLC-heteroduplex detection, thus 515 samples were screened for variance within cxon3-intron3-exon4. Exon 3

SSCP detection identified 20 positive band-shifts, 3 of which were due to sequence variance, while Exon 4 SSCP detection identified 28 positive band-shifts, 2 of which were due to sequence variance. Exon 3 SSCP detection failed to detect 1 sample with known sequence variance, as did Exon 4 SSCP detection. dHPLC-heteroduplex detection identified 25 positive peak-shifts, 2 of which were due to sequence variance.

Representative exon 3 SSCP detection results are shown in figure 2.4.2.6a, while representative exon 4 SSCP detection results are shown in figure 2.4.2.6b. Results later shown to correspond to a sequence change are shown in figures 2.4.2.6c (N125S), 2.4.2.6d (S170L), and 2.4.2.6e (525g>a). Representative dHPLC detection results are shown in figures 2.4.2.6f and 2.4.2.6g. Results later shown to correspond to a sequence change are shown in figures 2.4.2.6h (N125S), 2.4.2.6i (S170L), and 2.4.2.6j (525g>a). SSCP detection failed to detect 2 samples with sequence variance shown in figures 2.4.2.6m (EI49K) and 2.4.2.6e (525g>a).

• * v

*

(Figure 2.4.2.6a) A H E SX l exon 3 SSCP detection g e l show ing a sam ple with a SC C change (arrow) and a num ber o ffa ile d sam ples. This is an exam ple o f a f a ls e p ositive. Wild type is f a r right.

n i s ' i

i

L~' ^ ^

V ' . . . , " - y , . n ,