Prueba 5: Desempeño del controlador tipo PID y SMC en trayectoria

Table 3.5 shows a summary of the number of clonal sequences amplified by either DNA or RT-PCR. Amplification by RT-PCR using constant region primers proved to be more successful.

Table 3.5 Summary of PCR results

S s ? a ! f

Positive by DNA 29/49 (59%) Positive by RNA 11/12 (92%)

Total clones 40/49 (82%)

RT-PCR analysis was restricted by the availability o f tissue for RNA extraction

A total o f 77 rearrangements in 46 patients were identified by Southern Blotting analysis, although only 38 o f these were cloned. Twenty five out o f forty six patients showed bi- allelic rearrangements (presence o f 2 bands o f equal intensities), the most common reason for which is believed to be an unproductive D to J rearrangement on one allele leading to allelic exclusion followed by a productive rearrangement on the other. Although the JH probe is capable o f detecting unproductive rearrangements, they may not be amplifiable by PCR due to incomplete rearrangement o f V to the unproductive DJ. The failure to amplify more than one clone from those samples in which Southern

Blotting analysis showed 2 or more rearranged bands o f unequal intensity, suggesting oligoclonality rather than a bi-allelic clone, may be due to inefficient annealing o f the PCR primers due to mutations.

3.6 Sequence analysis

Immunoglobulin sequences were aligned to their germline counterparts and analysed for open reading frames, D N A and amino acid sequence hom ology to known VH genes, conserved splicing sequences and somatic hypermutations. Three out o f 40 sequences were found to be unproductive (ES, GM and JA) and are discussed separately in section 3.6.2. Table 3.6, Table 3.7 and Table 3.8 describe the IgH V, D and J segments used in each productive rearrangement, total numbers o f base pair differences compared to the most hom ologous germline VH sequence and the number o f amino acid changes compared to the germline protein sequence. The germline counterpart o f the Ig VH

segments could be identified in 37/37 productive sequences and Ig JH in 36/37 cases. Ig DH segments are difficult to align due their short sequence, the trimming and insertion of nucleotides by terminal deoxynucleotide transferase (TdT) at the VD and DJ junction and somatic hypermutation throughout the CDR3 region. The IMGT database recommends that Ig DH segments are checked manually in case of misalignment. Thirty out of thirty seven productive sequences could be assigned an Ig DH segment, the remaining 8 sequences could not be matched presumably due to a high somatic mutation rate.

Alignment to germline Ig VH segments allowed an accurate assessment o f the extent of somatic hypermutation (section 3.8)

An example of a rearranged sequence showing sites o f importance such as RNA splice sites, primer locations and framework and complementarity determining regions is shown in Figure 3.6. DNA and protein alignment to germline Ig VH segments with highlighted points of somatic hypermutation are shown for each productive sequence in Appendix 4.

Table 3.6 Ig gene segments used in the rearrangements: DLBL group Patient’s

initials

IgH gene segments VH gene

mutations'* Amino acid changes^ D^ J' DC 4-30.2 6-19 4 68 37 DD 3-30 2-15 3 23 12 FA 3-23 5-12 4 40 19 GK 5-51 ? 4 47 31 GS 3-23 1-14 1 41 19 ME 1-2 ? 4 28 17 M P 4-59 ? 5 19 13 JC 4-59 3-9/inv 6 11 7 EZ 4-59 2-21/inv 4 71 34 MH 4-59 1-? 4 32 19 JPF 3-23 3-3 4 24 11 JB 3-48 6-9 6 3 3 JAL 3-7 2-21/inv 4 24 12 M A 5-a 3-3 6 60 34

Table 3.7 Ig gene segments used in the rearrangments: FCL group P atient’s

initials

IgH gene segments VH gene

mutations^ Amino acid changes* V* D^ J' DT 3-23 4-23 5 59 23 JD 3-7 3-10 6 20 8 JR 3-7 ? 4 23 14 M M 4-34 ? ? 64 31 RH 3-11 6-19 5 47 20 SJ 3-9 ? 4 37 16 PR 1-2 5-18 3 6 4 SP 3-11 2-21 3 27 12 JF 3-23 2-2 4 40 21 TL 3-53 6-25 3 55 26

Table 3.8 Ig gene segments used in the rearrangements: Other lymphomas Patient’s

initials

IgH gene segments VH gene

mutations^ Amino acid changes* V* D^ j ' HAD 2-70 ? 5 * * WF 1-69 3-9 6 0 0 AS 3-30.5 3-3 4 18 14 JT 3-30 6-19 4 28 20 SS 3-33 2-21 6 12 10 BJ 3-66 6-13 4 3 1 GW 1-69 6-13 6 0 0 MB 1-69 3-3 6 1 1 HB 1-8 7-27 2 0 0 RB 1-8 6-19 6 2 1 JL 3-7 2-15 5 30 14 DB 3-30.3 6-19 4 6 5 UD 3-30.5 3-30.5 4 7 5 Tables 3.6, 3.7 and 3.8.

’ VH gene segment used in immunoglobulin gene rearrangement as determined by alignment to germline counterpart by IMGT/DNAPLOT

^ DH gene segment used, determined as above but checked manually

^ JH gene segment used, determined as above. VH genes could not be assigned for patient MM due to mutation throughout the JH gene segment.

Number of mutations in the VH gene segment from the beginning of FRl to the end of FR3

^ Number of amino acid changes within the VH gene segment from the beginning of FRl to the end of FR3, due to base pair mutations

* The sequence derived from patient HAD showed extensive intraclonal variation and numbers o f base pair and amino acid mutations could not be determined (see section 3.6.3 for detailed description)

Figure 3.6 Example of a rearranged sequence

This figure shows the DNA and protein sequence o f the immunoglobulin variable region from patient AS.

Key

Restriction sites are highlighted in blue

Single letter amino acid protein sequence is shown in purple, conserved residues are in bold and underlined.

Leader sequence is in two parts, separated by the intron and is double underlined RNA splice sites are boxed

5' VH leader and 3'JH primer sites are shown highlighted in yellow

VDJ coding sequence is shown in black upper case with FR and CDR regions indicated

The intronic sequence is shown in black lower case letters and separates the two parts o f the leader sequence

Figure 3.6 Example of a rearranged sequence

M E F G L S W V F L V A L L R

d o t l q a t t c a t q q a q a a a t a a a q a q a c t a a q t a t a a q t a a a c a t q a a t a a a a a a a a c t a a a t t t a t a t a a

C V Q L V E

c a t t t t c t g a t a a c g g t g c c c t t c t g t t t g c g o T GTC CAG TGT GAG GTG GAG GTG GTG GAG

< ---

V FRl

G G V y Ç P : S L R L S G A A

TGT GGG GGA GGG GTG GTG GAG GGT GGG AGG TGG GTG AGA GTG TGG TGT GGA GGG

>

G F T L F S Y G H H V R Q A P G

T G '^ G ( ^ TTG AGG GTG AGT AGT TAT GC^ A^G GAG TGG GTG GGG GAG GGT GGA GGG

V CDRl V FR2

G L E W V A I T p Y D G G K I Y Y

AAG GGG GTG GAG TGG GTG GGA GTT AGA GGA TAT GAT GGA GGG AAG ATA TAG TAT

> < > < ---

V CDR2

A E G y K G R F T I S R I S K N T

GGA GAG TGG GTG AAG GGG GGG TTT AGG ATG TGG AGA GAG ATT TGG AAG AAG AGG

>

V FR3

V L E M V s L w P E D T ' V Y Y G

GTG TAT GTG GAA ATG TAG AGG GTG AGA GGT GAG GAG AGG GGT GTG TAT TAG TGT

A K P F F P F S E S G N F F F D S W

GGG AAG GGG TTT GTA GGA TTT TGG GAG TGG GGA AAG TTG TTG TTT GAG TGT TGG

VDJ CDR3 J FR4

G Q G T L ' y T V S S

GGG GAG GGA AGG GTG GTG AGG GGT TGG TGA GGT GAATTG

>

3.6.1 Amplification of Ig VH sequences from a downstream JH segment

PCR amplification of DNA samples from patients PR, MH and JPF showed products of approximately 900 base pairs rather than the expected size o f -540 base pairs. The bands were excised, cloned and double strand sequenced.

Patient PR

Sequence analysis revealed a clonal VH rearrangement joined to a JH3 segment, however the 3' JH consensus primer annealed to the adjacent JH4 segment further downstream giving rise to a larger PCR product (Figure 3.7). This is in agreement with VH gene rearrangement whereby the DNA downstream of the recombined JH segment remains intact The most likely reason for the downstream JH primer annealing is that the JH3 has 3 mismatches with the JH consensus primer whereas JH4 is 100% homologous. Patients MH and JPF

Original PCR products for patients MH and JPF were also larger than expected and of similar size to that obtained for PR. However, following the digestion step prior to cloning, PCR products from patients MH and JPF were reduced to approximately 500 base pairs in size, suggesting the presence of an internal restriction site. Sequence analysis revealed clonal VH rearrangements in both samples and JH4 segment involvement. However, an internal EcoRI site had been generated within the JH4

segment, hence the shorter product following digestion (Figure 3.7). Amplification from a downstream JH segment is the most likely reason for the large PCR product, as shown in the analysis of the sequence from patient PR. The poor annealing of the JH primer to the JH4 segment involved in the rearrangement is likely to be due to somatic mutation, which also gave rise to an internal EcoRI site (not present in the germline JH sequence). We cannot, however, exclude the possibility that this could be an extremely rare

Figure 3.7 RT-PCR amplification of Ig VH on RNA

L l L2 V D J intron J intron

î

5 'V H l-6 family- EcoRI site 3 ' JH consensus

specific leader primers primer

The 3' JH consensus primer annealed to a downstream JH segment not involved in the rearrangement in 3 patient samples (PR, MH and JPF). In patients MH and JPF, an FcoRl site was introduced into the JH gene causing the PCR products to be digested during cloning.

3.6.2 Unproductive sequences

Unproductive clonal sequences were amplified from DNA samples from patients CM and FS. The VH4-34 sequence from patient GM contained a stop codon at triplet 77 in the 1 R3, resulting from a C—>T mutation. Southern Blotting analysis o f DNA from patient GM had previously shown two rearranged bands of equal intensity indicating a bi-allelic rearrangement. One allele is likely to be unproductive, while the other is productive and gives rise to the surface Ig identified by immunophenotyping. It is possible that the unproductive allele was amplified and cloned rather than the productive allele and this discrepancy could have been clarified by RT-PCR, had tissue been available.

The VH4-4 sequence from patient FS also showed a stop codon at triplet 51 in FR2, resulting from a G-^T mutation. This sequence also contains a frame shift that places D and J out of frame with V. The Southern Blotting results from patient FS showed only one rearranged band. Since immunophenotyping studies showed the presence of surface immunoglobulin, it is probable that PCR amplification introduced a mismatch into the sequence or that a technical error occuned during sequence analysis. Availability o f tissue for RT-PCR analysis may also have clarified this discrepancy.

The clonal sequence amplified by RT-PCR from patient JA was also unproductive due to an 80 base pair deletion covering FR2, CDR2 and part o f FR3. Southern Blotting results

showed that only one rearranged band existed and surface expression of immunoglobulin was positive by immunophenotyping. The reason for this large deletion is unknown.

3.6.3 Intraclonal variation

Sequence analysis on patient HAD revealed a clonal CDR3 region in which 5 out of 8 DNA samples sequenced in both directions were identical. However, there were a

multiple base pair changes scattered throughout the whole o f the VH sequence. This may have been the result of intraclonal variation where ongoing somatic hypermutation introduces changes into the clonal sequence. Patient HAD had an anaplastic large cell lymphoma (ALCL), a feature o f which is intraclonal variation