Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients
Deregulation of NOTCH1-signalling pathway is common in chronic lymphocytic leukemia (CLL). The most of studies are focused on detection of the hotspot c.7541_7542delCT NOTCH1 mutations in exon 34, while studies of mutations in the 3′UTR region are rare. The aims of work were to evaluate the frequenc...
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irk-123456789-1456362019-01-26T01:23:19Z Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients Abramenko, I.V. Bilous, N.I. Chumak, A.A. Dyagil, I.S. Martina, Z.V. Original contributions Deregulation of NOTCH1-signalling pathway is common in chronic lymphocytic leukemia (CLL). The most of studies are focused on detection of the hotspot c.7541_7542delCT NOTCH1 mutations in exon 34, while studies of mutations in the 3′UTR region are rare. The aims of work were to evaluate the frequencies of mutations in the 3′UTR region of the NOTCH1 gene (9:136,495553-136,495994) in Ukrainian CLL patients, the distribution of rs3124591 genotypes located in that area, and association of NOTCH1 mutations with structure of B-cell receptor. Materials and Methods: Detection of mutations in the 3′UTR region of the NOTCH1 was performed by direct sequencing in 87 previously untreated CLL patients (from the total group of 237 CLL patients) with unmutated immunoglobulin heavy-chain variable (UM IGHV) genes and without mutations in hotspot regions of TP53, SF3B1, and exon 34 of NOTCH1 genes. Results: Mutations in the 3′UTR region of the NOTCH1 were revealed in three of 87 CLL patients (3.4%). Two cases with non-coding mutations were related to subset #1 of stereotyped B-cell receptors, and one case belonged to stereotyped subset #28a. Analysis with inclusion of 30 UM IGHV cases with previously detected c.7544_7545delCT mutations revealed that the frequency of UM IGHV genes of I phylogenetic clan (except IGHV1-69) was significantly increased, and the frequency of UM IGHV3 and IGHV4 genes, on the contrary, was reduced in NOTCH1-mutated cases comparing with NOTCH1-unmutated cases (p = 0.002) and the general group (p = 0.013). SNP rs3124591 did not affect the risk of CLL and survival parameters of the patients. At the same time, differences were found in the frequency of IGHV gene usage and in the structure of HCDR3 in carriers of individual genotypes. Conclusion: The frequency of NOTCH1 mutations in 3′UTR region was low. Our findings confirmed current data on the association between the structure of the B-cell receptor and the appearance of NOTCH1 mutations. Some features of HCDR3 structure were identified in carriers of TT and CC genotypes of rs3124591. Key Words: NOTCH1 mutations, 3′UTR region of the NOTCH1, rs3124591, IGHV genes. 2018 Article Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients / I.V. Abramenko, N.I. Bilous, A.A. Chumak, I.S. Dyagil, Z.V. Martina // Experimental Oncology. — 2018 — Т. 40, № 3. — С. 211-217. — Бібліогр.: 31 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145636 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Abramenko, I.V. Bilous, N.I. Chumak, A.A. Dyagil, I.S. Martina, Z.V. Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients Experimental Oncology |
| description |
Deregulation of NOTCH1-signalling pathway is common in chronic lymphocytic leukemia (CLL). The most of studies are focused on detection of the hotspot c.7541_7542delCT NOTCH1 mutations in exon 34, while studies of mutations in the 3′UTR region are rare. The aims of work were to evaluate the frequencies of mutations in the 3′UTR region of the NOTCH1 gene (9:136,495553-136,495994) in Ukrainian CLL patients, the distribution of rs3124591 genotypes located in that area, and association of NOTCH1 mutations with structure of B-cell receptor. Materials and Methods: Detection of mutations in the 3′UTR region of the NOTCH1 was performed by direct sequencing in 87 previously untreated CLL patients (from the total group of 237 CLL patients) with unmutated immunoglobulin heavy-chain variable (UM IGHV) genes and without mutations in hotspot regions of TP53, SF3B1, and exon 34 of NOTCH1 genes. Results: Mutations in the 3′UTR region of the NOTCH1 were revealed in three of 87 CLL patients (3.4%). Two cases with non-coding mutations were related to subset #1 of stereotyped B-cell receptors, and one case belonged to stereotyped subset #28a. Analysis with inclusion of 30 UM IGHV cases with previously detected c.7544_7545delCT mutations revealed that the frequency of UM IGHV genes of I phylogenetic clan (except IGHV1-69) was significantly increased, and the frequency of UM IGHV3 and IGHV4 genes, on the contrary, was reduced in NOTCH1-mutated cases comparing with NOTCH1-unmutated cases (p = 0.002) and the general group (p = 0.013). SNP rs3124591 did not affect the risk of CLL and survival parameters of the patients. At the same time, differences were found in the frequency of IGHV gene usage and in the structure of HCDR3 in carriers of individual genotypes. Conclusion: The frequency of NOTCH1 mutations in 3′UTR region was low. Our findings confirmed current data on the association between the structure of the B-cell receptor and the appearance of NOTCH1 mutations. Some features of HCDR3 structure were identified in carriers of TT and CC genotypes of rs3124591. Key Words: NOTCH1 mutations, 3′UTR region of the NOTCH1, rs3124591, IGHV genes. |
| format |
Article |
| author |
Abramenko, I.V. Bilous, N.I. Chumak, A.A. Dyagil, I.S. Martina, Z.V. |
| author_facet |
Abramenko, I.V. Bilous, N.I. Chumak, A.A. Dyagil, I.S. Martina, Z.V. |
| author_sort |
Abramenko, I.V. |
| title |
Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients |
| title_short |
Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients |
| title_full |
Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients |
| title_fullStr |
Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients |
| title_full_unstemmed |
Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients |
| title_sort |
analysis of the 3′utr region of the notch1 gene in chronic lymphocytic leukemia patients |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2018 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/145636 |
| citation_txt |
Analysis of the 3′UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients / I.V. Abramenko, N.I. Bilous, A.A. Chumak, I.S. Dyagil, Z.V. Martina // Experimental Oncology. — 2018 — Т. 40, № 3. — С. 211-217. — Бібліогр.: 31 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT abramenkoiv analysisofthe3utrregionofthenotch1geneinchroniclymphocyticleukemiapatients AT bilousni analysisofthe3utrregionofthenotch1geneinchroniclymphocyticleukemiapatients AT chumakaa analysisofthe3utrregionofthenotch1geneinchroniclymphocyticleukemiapatients AT dyagilis analysisofthe3utrregionofthenotch1geneinchroniclymphocyticleukemiapatients AT martinazv analysisofthe3utrregionofthenotch1geneinchroniclymphocyticleukemiapatients |
| first_indexed |
2025-07-10T22:08:22Z |
| last_indexed |
2025-07-10T22:08:22Z |
| _version_ |
1837299484641460224 |
| fulltext |
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ���
ANALYSIS OF THE 3΄UTR REGION OF THE NOTCH1 GENE
IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS
I.V. Abramenko1, *, N.I. Bilous1, A.A. Chumak1, I.S. Dyagil2, Z.V. Martina2
1Department of Clinical Immunology, Research Center for Radiation Medicine, Kyiv 02000, Ukraine
2Department of Hematology, Research Center for Radiation Medicine, Kyiv 02000, Ukraine
Deregulation of NOTCH1-signalling pathway is common in chronic lymphocytic leukemia (CLL). The most of studies are fo-
cused on detection of the hotspot c.7541_7542delCT NOTCH1 mutations in exon 34, while studies of mutations in the 3′UTR
region are rare. The aims of work were to evaluate the frequencies of mutations in the 3′UTR region of the NOTCH1 gene
(9:136,495553-136,495994) in Ukrainian CLL patients, the distribution of rs3124591 genotypes located in that area, and associa-
tion of NOTCH1 mutations with structure of B-cell receptor. Materials and Methods: Detection of mutations in the 3′UTR region
of the NOTCH1 was performed by direct sequencing in 87 previously untreated CLL patients (from the total group of 237 CLL
patients) with unmutated immunoglobulin heavy-chain variable (UM IGHV) genes and without mutations in hotspot regions of TP53,
SF3B1, and exon 34 of NOTCH1 genes. Results: Mutations in the 3′UTR region of the NOTCH1 were revealed in three of 87 CLL
patients (3.4%). Two cases with non-coding mutations were related to subset #1 of stereotyped B-cell receptors, and one case
belonged to stereotyped subset #28a. Analysis with inclusion of 30 UM IGHV cases with previously detected c.7544_7545delCT
mutations revealed that the frequency of UM IGHV genes of I phylogenetic clan (except IGHV1-69) was significantly increased,
and the frequency of UM IGHV3 and IGHV4 genes, on the contrary, was reduced in NOTCH1-mutated cases comparing with
NOTCH1-unmutated cases (p = 0.002) and the general group (p = 0.013). SNP rs3124591 did not affect the risk of CLL and
survival parameters of the patients. At the same time, differences were found in the frequency of IGHV gene usage and in the
structure of HCDR3 in carriers of individual genotypes. Conclusion: The frequency of NOTCH1 mutations in 3′UTR region was
low. Our findings confirmed current data on the association between the structure of the B-cell receptor and the appearance
of NOTCH1 mutations. Some features of HCDR3 structure were identified in carriers of TT and CC genotypes of rs3124591.
Key Words: NOTCH1 mutations, 3′UTR region of the NOTCH1, rs3124591, IGHV genes.
Deregulation of NOTCH1-signalling pathway
is common in chronic lymphocytic leukemia �CLL�.
This gene encodes transmem�rane receptor� and
active intracellular fragment of NOTCH� �ICN�� re-
leasing after ligand �inding� conformation changes
and proteolytic cleavages translocates to the nu-
cleus� where it mediates the transcription of target
genes involved in cell differentiation� proliferation
and apoptosis [�� �]. Expression of ICN� was found
in 5�.5% of CLL cases lacking NOTCH1 gene muta-
tions regardless of mutation status of immunoglo�ulin
heavy-chain varia�le �IGHV� genes [3]. Prognostic
significance of such constitutional ICN� expression
in CLL is not yet known.
Activated NOTCH1 mutations in CLL were first found
through whole-genome sequencing �y two independent
groups in ���� [�� 5]. They were commonly represented
�y a single �-�p deletion �c.�5��_�5�5delCT� P�5��fs�
in exon 3� and resulted in the removal of C-terminal
PE�T [proline �P�� glutamic acid �E�� serine ���� and
threonine �T� rich] domain involved in proteasomal
degradation of ICN�. NOTCH1 c.�5��_�5�5delCT
mutations occur in ����% in newly diagnosed CLL�
����5% at the time of first treatment and �5���%
at chemorefractoriness and� according to the data
of several authors� are associated with short progres-
sive-free �PF�� and overall survival �O�� [6� �]. Later�
in the minority of CLL cases point mutations in the
3´UTR region of NOTCH1 were found that resulted in the
removal of C-terminal PE�T domain [�]. We revealed
c.�5��_�5�5delCT mutations of NOTCH1 in �3.�%
of untreated CLL patients. Associations �etween the
presence of NOTCH1 mutations and unmutated �UM�
IGHV genes� more advanced stage of the disease�
higher initial WBC count� �ulky disease� short time-to-
treatment �TTT� period and PF� were found [9]. The
aims of present work were to evaluate the frequencies
of mutations in the 3´UTR region of the NOTCH1 gene
�9:�36��95553��36��9599�� in Ukrainian CLL patients�
the distri�ution of rs3���59� genotypes located in that
area� and association of NOTCH1 mutations with struc-
ture of B-cell receptor.
MATERIALS AND METHODS
�amples of �� previously untreated CLL patients
with UM IGHV genes and without mutations in hotspot
regions of TP53� SF3B1, and NOTCH1 �exon 3��
genes were selected for the study from the total
group of �3� patients. �uch choice of patients was
�ased on the current data on a rare association
of the NOTCH1 mutations with mutations of TP53 and
SF3B1 genes� and their prevalence in cases with
UM IGHV genes. All patients �69 males and �� females�
were referred to the National Research Center for Ra-
diation Medicine� Kyiv� during the period of ��������6.
The study was approved �y the local Ethics Review
Exp Oncol ����
��� 3� �������
Submitted: May 29, 2018.
*Correspondence: E-mail: abramenko_iryna@ukr.net
Abbreviations used: CLL — chronic lymphocytic leukemia;
HCDR3 — heavy chain complementarity-determining region
3; ICN1 — intracellular fragment of NOTCH1; IGHV — immuno-
globulin heavy-chain variable (genes); OS — overall survival;
PFS — progression-free survival; TTT — time-to-treatment; UM —
unmutated.
��� Experimental Oncology ��� �������� ���� ��eptem�er�
Committee� and all patients gave informed consent
prior to participation in it. The diagnosis of CLL was
�ased on clinical history� lymphocyte morphology� and
immunophenotypic criteria.
Genomic DNA for molecular analysis was ex-
tracted from peripheral �lood mononuclear cells with
the QIAamp Blood Mini Kit �Qiagen� Crawley� United
Kingdom� according to the manufacturer’s protocol.
�creening for presence of TP53� SF3B1 mutations and
NOTCH1 mutations in exon 3� as well as mutational
status of IGHV genes was performed in all �3� patients.
IGHV-D-J rearrangements were amplified ac-
cording to the BIOMED-� consortium rules [��]
as descri�ed previously [��]. IGHV rearrangements
were analyzed �y IMGT/V-QUE�T [��]. Mutational
status of TP53 gene was performed for exons
3 to �� as descri�ed previously [�3]. SF3B1 muta-
tions were analyzed in exons ��� �5 and �6 �y PCR
amplification followed �y direct sequencing with the
BigDye Terminator v3.� Cycle �equencing kit �Applied
Biosystems� according to Rossi et al. [��]. It should
�e noted that sequencing was carried out in those
regions of TP53 and SF3B1 genes where the vast
majority of mutations is located �> 95%� [�5� �6].
NOTCH1 mutations were analyzed in the hotspot
c.����_�6�� region in exon 3� of NOTCH1 gene
�y PCR amplification followed �y direct sequencing
as descri�ed previously [9]. Detection of mutations
in the 3´UTR region of NOTCH1 gene �9:�36��95553�
�36��9599�� was performed �y direct sequencing
according to Puente et al. [�] using original prim-
ers. PCR conditions and primers are descri�ed in:
dx.doi.org/��.��5��/protocols.io.qfhdtj6.
Analysis of rs3���59� genotypes was performed
using �NPstats tool �http://�ioinfo.iconcologia.net/
snpstats/start.htm�.
TTT period� PF� and O� were estimated �y the
method of Kaplan and Meier and assessed �y the
log-rank test.
RESULTS
The frequency of NOTCH1 mutations and as-
sociation with HCDR3 structure. Mutations in the
3´UTR region of the NOTCH1 were revealed in three
of �� CLL patients �3.�%�. Non-coding mutations
were represented �y �3939��5�T>C (two cases) and
�3939���5T>C �one case�.
Two cases with non-coding mutations were related
to su�set #� of stereotyped B-cell receptors� and one
case �elonged to stereotyped su�set #��a �according
to classifications of �tamatopoulos et al. [��]� Murray
et al. [��]� Bom�en et al. [�9]�.
To study the associations of NOTCH1 mutations
and the stereotyped B-cell receptors� we have added
to the studied group 3� UM IGHV cases with previ-
ously revealed c.�5��_�5�5delCT mutations from
the total group of �3� CLL patients. Thus� com�ined
group of ��� UM-IGHV cases included 33 cases
with NOTCH1 mutations and �� cases without
NOTCH1 mutations in exon 3� and in the 3´UTR region.
It was divided into three su�groups according to the
principle used �y �utton et al. [��].
The frequency of NOTCH1 mutations in su�group
of �� CLL cases with UM IGHV genes of I phylogenetic
clan �comprising IGHV1, IGHV5 and IGHV7 genes��
except IGHV1-69� was the highest ��3 of �� cases;
59.�%�. This su�group included 9 cases of su�set
#� �five with NOTCH1 mutations�� � cases of su�set
#��a ��oth NOTCH1-mutated�� one case of su�sets
#59� #95� #UA/ref� [��] �all NOTCH1-mutated�� six
cases showing homology with CLL sequences rep-
resented in the data�ases� �ut did not include in the
stereotyped su�sets �three with NOTCH1 mutations��
and two NOTCH1-UM heterogeneous cases. Lengths
of HCDR3 in NOTCH1-mutated cases varied from �� a.a.
to �� a.a. and did not differ from NOTCH1-UM cases
��5.66 ± �.9� a.a. vs ��.55 ± �.93 a.a.; p = �.�65�.
Additional NOTCH1-mutated CLL case� not includ-
ed in this su�group� in its structure of IGHV-D-J rear-
rangement was close to cases of su�sets #� �struc-
tures of HCDR3 in CLL cases with NOTCH1 mutations
are availa�le �y request�.
In su�group of 3� CLL cases with UM IGHV1-69 gene�
NOTCH1 mutations were revealed in �� cases �3�.�%�.
NOTCH1-mutated cases �elonged to su�sets #6 �� of 3��
#9 �� of 3�� #UA/ref� �� of ��� #UA/ref�� �one case�.
Additional one NOTCH1-mutated case was revealed
among six cases homologous with CLL sequences
represented in the data�ases� �ut did not include in the
stereotyped su�types� and seven NOTCH1 mutations
were found among fourteen CLL cases with heteroge-
neous HCDR3s. Without NOTCH1 mutations there were
cases of su�sets #� �n = 5�� #UA� �n = ��� #UA6 �n = ���
and #UA� �n = ��. Lengths of HCDR3 in NOTCH1-
mutated cases varied from �� a.a. to �5 a.a. and did
not differ from NOTCH1-UM cases ���.�� ± �.�5 a.a.
vs ��.6� ± �.�� a.a.; p = �.635�.
In su�group of 5� CLL cases with other UM genes
�IGHV3� n = 39; IGHV4� n = �6; IGHV2� n = �� NOTCH1 mu-
tations were present in seven cases ���.3%�. Two
NOTCH1-mutated cases �elonged to su�sets
#�� �� of 3� and #�5 �one case�� two had homology
with CLL sequences represented in the data�ases�
�ut not included in the stereotyped su�types �total
n = �5�� the three remaining cases had heteroge-
neous HCDR3s �total n = �5�. CLL cases from su�sets
#� �n = ��� #9 �n = ��� #3� �n = ��� #�� �n = ��� #�� �n = ���
#5� �n = ��� #��9 �n = ��� #UA5 �n = ��� and #UA9 �n = ��
were NOTCH1-UM. Mean lengths of HCDR3 did not
differ in NOTCH1-mutated ���.�� ± �.9� a.a.� and
NOTCH1-UM cases ���.6� ± �.6� a.a.; p = �.�6��.
Differences in the frequency of NOTCH1 mutations
in su�groups of cases expressed UM IGHV genes
of I clan �except IGHV1-69�� IGHV1-69 gene and
other UM IGHV genes were significant �p = �.����.
The distri�ution of UM IGHV genes of the selected
su�groups among NOTCH1-mutated cases was
significantly different comparing with the distri�u-
tion in NOTCH1-UM cases� in the analyzed group
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ��3
of ��� CLL cases as well as in total group of �3� CLL
cases �Ta�le ��.
Table 1. The distribution of UM IGHV genes in studied CLL patients
Groups of CLL patients
Subgroups of UM IGHV genes p value
in compa-
rison with
NOTCH1-
mutated
cases
Clan
I except
IGHV1-69
IGHV1-69
Other IGHV
genes, in-
cluding
IGHV6-1
case
NOTCH1-mutated, n = 33 13 (39.4) 12 (36.4) 8 (24.2) –
NOTCH1-UM, n = 84 9 (10.8) 25 (27.7) 50 (59.5) 0.002
Whole group
of 117 patients
22 (18.8) 37 (31.6) 58 (49.6) 0.013
161 cases with UM IGHV
genes from total group
of 237 patients
32 (19.9) 50 (31.0) 79 (49.1) 0.013
Analysis of rs3124591 SNP distribution and its as-
sociation with survival parameters and IGHV gene us-
age. The distri�ution of rs3���59� genotypes was as fol-
lows: CC genotype — �� cases ���.5%�� CT genotype —
�� cases ���.�%�� and TT genotype — 3� cases �3�.�%�
and did not deviate from the Hardy — Wein�erg equili�rium
�p = �.���. In comparison with healthy individuals of Eu-
ropean ancestry retrieved from the ���� Genomes Proj-
ect dataset �CC genotype �6.�%; CT genotype ��.�%;
TT genotype �6.�%; http://www.����genomes.org/�
no significant differences were found. All three cases with
non-coding NOTCH1 mutations were represented in car-
riers of TT genotype.
The impact of rs3���59� on duration of TTT period�
PF� and O� was insignificant �p = �.��5� p = �.3���
and p = �.���� correspondingly�.
The spectrum of used IGHV genes tended
to �e narrower in CC homozygotes than in carriers
of CT and TT genotypes �correspondingly ��� ���
and � IGHV genes� p = �.��5; p = �.��3 in comparison
TT vs CC genotype carriers� �Ta�le ��.
Table 2. The distribution of UM IGHV genes in carriers of different
rs3124591 genotypes
IGHV genes Genotypes of rs3124591
CC, n = 19 CT, n = 37 TT, n = 31
IGHV1 family 9 (47.4) 17 (45.9) 9 (29.0)
IGHV1-69 8 (42.1) 13 (35.1) 4 (12.9)
IGHV2 family 0 0 1 (3.2)
IGHV3 family 7 (36.8) 13 (35.1) 14 (45.2)
IGHV3-11 4 (21.1) 1 (2.7) 2 (6.5)
IGHV4 family 3 (15.0) 7 (18.9) 4 (12.9)
IGHV4-39 2 (10.5) 2 (5.4) 0
IGHV5 family 0 0 3 (9.7)
Expression of only two IGHV genes was detected
in more than half of CC homozygotes �� cases with
IGHV1-69 and � cases with IGHV3-11 gene�.We found
a reduced IGHV1-69 gene usage in carriers of TT geno-
type compared to carriers of CT and CC genotypes
���.9%� 35.�%� and ��.�%� correspondingly;
p = �.�3��. The distri�utions of IGHD and IGHJ genes
were compara�le in carriers of different genotypes.
It should �e noted that the frequencies of IGHV and
IGHJ genes usage and different stereotyped su�sets
in this studied group did not differ from those in the
previously studied large group cohort.
The HCDR3 length did not differ in carriers of dif-
ferent genotypes with UM IGHV genes �CC genotype
��.�3 ± �.�� a.a.; CT genotype ��.�5 ± �.�3 a.a.;
TT genotype �9.�6 ± �.�6 a.a.; p = �.����. However� the
num�er of N nucleotides inserted in the VHD junctions
was significantly less in carriers of TT genotype than
in carriers of CT and CC genotypes �Ta�le 3�.
The comparison of CLL sequences with non-CLL
sequences availa�le from pu�lic data�ases showed
that most cases that had HCDR3 homology with anti-
�acterial or antiviral Ig clones were present in TT homo-
zygotes ���.6% vs �.�% in carriers of CT genotype and
�% in carriers of CC genotype� p = �.��6; Ta�le ��. All
CLL sequences homologous with autoreactive clones
were revealed in carriers of TT �9.�%� and CT ���.�%�
genotypes. A num�er of CLL cases that were similar
to Ig sequences expressed �y normal B-cells �elderly�
neonate� cord �lood� tonsils� and to Ig sequences from
patients with X-linked hyperIgM did not differ in carriers
of different rs3���59� genotypes �Ta�le 5� 6�.
Table 4. HCDR3 homology in carriers of different rs3124591 genotypes
HCDR3 homology with: Genotypes p valueCC CT TT
Normal B cells 4 (21.1) 10 (27.0) 5 (16.1) 0.515
Ig from X-HIgM syndrome 0 1 (2.7) 3 (9.7) 0.226
Antibacterial or antiviral Ig clones 0 1 (2.7) 7 (22.6) 0.006
Autoreactive clones 0 4 (10.8) 3 (9.7) 0.332
DISCUSSION
To investigate non-coding mutations in the
3´UTR region of the NOTCH1� previously untreated
CLL cases with no mutations in TP53� SF3B1,
and NOTCH1 �exon 3�� genes were selected. All
Table 3. N-nucleotide additions and exonuclease activities in the VHD and DJH junctions in carriers of different rs3124591 genotypes
Parameters Genotypes p valueCC CT TT
VHD N-nucleotide addition (bp) 8.68 ± 1.05 9.33 ± 1.01 5.56 ± 0.83 0.014
VHD with no addition (% of sequences) 0 8.1 16.1 0.144
VHD N-nucleotide addition (bp) except cases with no addition 8.68 ± 1.05 10.18 ± 0.96 6.68 ± 0.83 0.032
DJH N-nucleotide addition (bp) 10.21 ± 2.19 7.72 ± 0.95 8.56 ± 1.10 0.443
DJH with no addition (% of sequences) 0 10.8 3.22 0.190
DJH N-nucleotide addition (bp) except cases with no addition 10.21 ± 2.19 8.68 ± 0.94 8.86 ± 1.10 0.716
VH 3' coding end excision (bp) 1.56 ± 0.19 1.63 ± 0.25 1.73 ± 0.43 0.542
VH 3' without excision (% of sequences) 36.8 27.03 38.71 0.415
VH 3' coding end excision (bp) except cases without excision 1.83 ± 0.29 2.26 ± 0.27 3.05 ± 0.60 0.157
D 5' coding end excision (bp) 5.11 ± 1.10 4.33 ± 0.61 4.23 ± 0.85 0.762
D 5' without excision (% of sequences) 0 16.2 25.8 0.049
D 5' coding end excision (bp) except cases without excision 5.11 ± 1.10 5.20 ± 0.62 5.77 ± 0.96 0.848
D 3' coding end excision (bp) 5.05 ± 0.77 5.00 ± 0.71 3.73 ± 0.72 0.365
D 3' without excision (% of sequences) 15.8 18.9 22.6 0.808
D 3' coding end excision (bp) except cases without excision 6.00 ± 0.68 6.21 ± 0.72 4.86 ± 0.79 0.395
JH 5' coding end excision (bp) 3.00 ± 0.87 3.66 ± 0.59 4.10 ± 0.64 0.581
JH 5' without excision (% of sequences) 26.3 27.03 22.58 0.918
JH 5' coding end excision (bp) except cases without excision 4.07 ± 1.04 5.07 ± 0.62 5.34 ± 0.62 0.510
��� Experimental Oncology ��� �������� ���� ��eptem�er�
of them expressed UM IGHV genes. �uch choice
was due to the literature data on a rare association
of the NOTCH1 mutations with mutations of the other
genes. In addition� NOTCH1 mutations are mainly
detected in CLL patients with UM IGHV genes. For
example� in the study of Rossi et al. �6.5% of CLL
cases with NOTCH1 mutations in exon 3� expressed
UM IGHV genes� and more than 9�% of them did
not have mutations of TP53 gene [6]. In the study
of �chnaiter et al.� in none of 9� fludara�ine-refractory
CLL patients concurrent NOTCH1 and SF3B1 were
found� �ut �3.�% of NOTCH1-mutated cases had
simultaneously TP53 gene mutations. All revealed
NOTCH1 mutations in this study were in patients with
UM IGHV genes [��]. In our group of �3� CLL patients�
mutations of TP53� SF3B1� and c.�5��_�5�5delCT
of NOTCH1 were found in ��.�%� ��.�%� and �3.�%
of cases� correspondingly. Only single case har�ored
mutations in �oth NOTCH1 and TP53 genes and two
cases — in NOTCH1 and SF3B1 gene. All except
one NOTCH1-mutated cases had UM IGHV genes.
Therefore� we investigated a group of CLL patients
with an increased chance to identify mutations in the
3´UTR region of NOTCH1 gene.
Non-coding mutations in the 3´UTR region
of NOTCH1 gene were first identified �y Puente
et al. [�]. Authors revealed �� NOTCH1 mutated cases
of ��6 CLL patients with UM IGHV genes �6.�%�. The
most frequent recurrent non-coding mutation was
�3939��5�T>C� and two cases had �3939���5 and
�3939���3 point mutations. Nadeu et al. detected
3´UTR mutations in �� of 39� CLL patients �5.6%� [�3].
We have identified two cases with �3939��5�T>C muta-
tion and one case with �3939���5T>C mutation among
�� IGHV-UM CLL cases �3.�%�. The frequency of non-
coding mutations in our group and their localization
were compara�le with previously pu�lished data.
Our data confirmed current data on the associa-
tion �etween the structure of the B-cell receptor and
appearance of NOTCH1 mutations. In one of the larg-
est series of studied stereotyped su�set CLL cases
�565 cases assigned to one of �� major stereotyped
su�sets� the skewed distri�ution of NOTCH1 muta-
tions within exon 3� was found. Enriched mutations
were cases �elonged to su�sets #�� #59� #99� #6�
and #�. NOTCH1 mutations were relatively infrequent
in cases from su�sets #3� #5� #� and� especially� from
su�sets #� and #� [��]. In our group� five of the nine
cases from stereotyped su�set #� were NOTCH1-
mutated: three had mutations within exon 3� and
two had non-coding mutations in the 3´UTR region.
On the contrary� NOTCH1 mutations were not found
among nine cases of su�set #�. The num�er of cases
�elonged to other su�sets was too low for comparison.
However� the o�served differences in the frequency
of NOTCH1 mutations in su�groups of cases that
expressed UM IGHV genes of I clan �except IGHV1-
69)� UM IGHV1-69 gene and other UM IGHV genes
�59.�%� 3�.�%� ��.3%� correspondingly� cannot
�e explained solely �y the presence in the first su�-
group of the cases �elonged to su�set #�. Excluding
cases of su�set #�� NOTCH1 mutations were detected
in eight of �� remaining cases of the first su�group
���.�%�. Both cases of su�set #��a were NOTCH1-
Table 5. Structure of HCDR3 region in CLL cases with NOTCH1 mutations (MAS — mean alignment score)
CLL cases HCDR3 amino acid sequence Subset
1-03*01_6-19*01_4*02_UM_EF407847 AREQWLGPSYFDY 1
1-02*02_6-19*01_4*02_UM_EU350413 ARAQWLVQLSLDY 1
1-02*02_6-19*01_4*02_UM_S42 ARLQWLWPRKLDY 1
5a*01_3-22*-1_4*02_UM_EF441753 ARIQWLLLPHFDY 1
5a*03_3-16*-1_4*02_UM_KC802107 ARLQFLGISDPFDY 1
1-02*02_1-26*01_6*02_UM_EF091912 ARPYSGSYPWYYYGMDV 28a
1-02*02_3-10*01_6*02_UM_EU667602 ARLYSGSYFYYYYGMDV 28a
1-58*01_3-3*01_6*02_UM_EF091913 AAGYDFWSGMDV 59
1-02*02_3-22*01_4*02_UM_GU358667 ERSYDSSGYYCHFDY 95
1-58*01_6-13*01_3*02_UM_EF175413 ALASSWIFDAFDI UA/ref4
1-02*04_2-8*01_6*02_UM_EU433868 AKPSFYCTNGVCYTDYYYGMDV Homology with CLL cases DQ100687 (MAS 68.2), EF177969 (MAS 68.2)
1-46*01_3-9*01_5*02_UM_GU358664 ARDRGYFDWLLRNGWFDP Homology with CLL case EF441746 (MAS 72.2)
5-51*01_3-3*01_4*02_UM_JQ928948 ARHGMYYDFWSGYYLAAYFDY Homology with CLL cases EU099117 (MAS 66.6); DQ100922 (MAS 61.9)
6-1*01_6-19*01_4*02_UM_JF810281 ARDEYWGSGWDY
1-69*06_3-16*02_3*02_UM_GU358670 ARGGDYDYIWGSYRSNDAFDI 6
1-69*01_3-3*01_6*02_UM_EF407834 ASKSLPITIFGVVISDYYYYGMDV 9
1-69*01_6-13*01_6*02_UM_EF175412 ARVQGGSAAAYENYYYYYGMDV UA/ref2
1-69*01_2-2*02_5*02_UM_EU350410 AREFSDIVVVPAAIIRNWFDP UA/ref11
1-69*01_3-3*01_6*02_UM_EU667595 ARAPDFWSGYYFRGGGMDV Homology with CLL case DQ100846 (MAS 68.4)
1-69*01_2-2*02_6*01_UM_EU433873 AREGGDIVVVPAAIISWSRYYGMDV
1-69*01_3-22*01_4*02_UM_S9 ARWGGGAYYYDSSGYYYGFDYYFDY
1-69*01_3-22*01_5*02_UM_KC02101 ARRNSGYYYYKEYNWFDP
1-69*01_3-9*01_4*02_UM_EF441755 ARDSRELRYFDWLSQEGYFDY
1-69*13_5-24*01_6*02_UM_GU358657 AREGDGYNYGYYYYYGMDV
1-69*01_6-19*01_6*03_UM_JX462742 ARVGGYSSGWYQNYYYYYMDV
1-69*01_3-9*01_4*02_UM_HM173329 VRMHFDWLRPAFYSFDY
3-11*01_3-3*01_6*02_UM_EF175391 ARTYYDFWSGYDGHYGMDV 22
3-30*03_3-3*01_6*02_UM_EU814961 AKDGLGIRFLEWLSTSYYYGMDV 25
4-04*02_2-2*01_5*02_UM_EU350427 ARVPIVVVPAAVSLMRVNWFDP Homology with CLL case EU099080 (MAS 77.2)
4-34*01_6-6*01_6*03_UM_GU358661 ARGLTYSSSSYYYYYYMDV Homology with CLL case EF407849 (MAS 68.4)
3-53*01_3-3*01_6*02_UM_EF175410 ARDASPSLYYDFYPYGMDV
3-64*05_6_19*01_2*01_UM_HM173330 VKDSTPGIAVAGTWGYWYFDL
2-70*01_3-3*01_4*02_UM_S10 ARTTKLSVYDFWSGYYTGSGLGYFDY
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ��5
Table 6. CLL cases showing HCDR3 homology with immunoglobulins of known specificity (MAS — mean alignment score)
IGHV gene HCDR3 amino acid sequence MAS Subset Antigen specificity
CC genotype
3-11*01_3-3*01_6*02_UM_LM647765 ARA--YDFWSGY-YFERYGMDV Peripheral blood B lymphocyte
3-11*01_3-3*02_6*02_UM_JQ928963 ARWGPYDFWSGYSYYYYYGMDV 68.1 22
1-69*01_2-2*01_6*02_UM_AB204177 AR_GGYCSSTSCTILSYYYYGMDV Umbilical cord blood CD19+IgD+CD27-CD38+
B lymphocyte
1-69*01_2-2*02_6*04_UM_JX462747 ARWSGYCSSTSCMGADYYYYGMDV 75.0
4-39*01_3-10*01_5*02_UM_AY607328 ARRLSYYYGSGSYYNWFDP Term cord blood
3-11*01_3-10*01_5*02_UM_JQ928962 ARDNVLYYGSGSYFNWFDP 73.0
4-34*01_3-3*01_6*03_UM_LM647851 ARGRVGYDFWSGS-PYYYYYYMDV Peripheral blood B lymphocyte
1-69*01_3-3*01_6*03_UM_KC802110 ARGRN-YDFWSGPTWGYYYYYMDV 73.0 7
TC genotype KC802096
4-34*01_2-2*02_4*02_UM_AY607318 ARTIIVVVPAAIRWFDP Term cord blood
4-34*01_2-2*02_4*02_UM_KC802096 AREDIVVVPAALYYFDY 70.6
3-11*04_3-10*01_4*02_M_AB202917 ARDSLWFGEFMY--FDY Tonsillar B cell
3-11*01_3-10*01_4*02_UM_EU433870 ARDTLWFGEFHAYYFDY 70.6 UA/ITA_13
1-8*01_-0*01_3*02_UM_EU571887 ASVLGTYYYGSGSYYDAFDI IgM+ B lymphocyte 18 week gestation fetus
spleen
4-30-4*01_3-0*01_3*02_UM_GU358680 ARK--TYYYGSGSYYDAFDI 77.7
6-1*01_6-6*03_6*03_UM_LM648697 AREGSSWSGNYYYYYYMDV Peripheral blood B cell
3-49*03_6-13*01_6*-3_UM_EF175429 TSHSSSWDYYYYYYYYMDV 63.1
3-33*01_6-19*01_6*02_UM_AF174112 ARDRLAVAGTVYYYYGMDV Elderly B cell
3-33*01_4-11*01_6*02_UM_EF407833 ARDLHAVTTRNYYYYGMDV 68.4 44
3-07*1_3-22*01_6*01_UM_DQ454347 ARV-GDYYDSSGYYYYYYGMDV Neonate peripheral blood
4-59*_3-22*01_6*02_UM_GU358662 ARGLGDYYDSSGYLHYYYGMDV 81.8 50
3-33*01_3-3*01_6*02_UM_AY607518 ARDTPY-DFWSGYYYYYYGMDV Term cord blood
3-33*01_3-3*01_6*02_UM_JX462745 ARDTRVDDFWSGYFVYYYGMDV 77.0 22
3-30*03_3-22*01_6*02_UM_LM648585 ARDWASRDSSGYYYL-GYYYYMDV Peripheral blood B cell
3-07*1_3-22*01_6*02_UM_EU814965 ARDTYYYDSSGYYYPYYYYYGMDV 65.0
1-69*01_2-2*01_6*02_UM_LM648973 ARDS--DIVVVPAARGPYYYGMDV Peripheral blood B cell
1-69*01_2-2*01_6*02_UM_EF091906 ASPGPQDIVVVPAAYYYYYYGMDV 79.0
1-8*01_2-2*01_6*02_UM_AJ414783 AR--VTGYCSSTSCTKVYYYYYGMDV B cell
3-21*-1_2-2*01_6*02_UM_EF091902 ARNRYTEYCSSTSCHPSYYYYYGMDV 73.0 41
3-33*01_3-3*01_6*02_UM_EF541600 ARDISTDFWSGYYT--GSYYYGMDVW Ig in X-HIgM Syndrome patient
1-2*01_3-3*01_6*02_UM_EU667601 ARGVSYDFWSGYYIREGDYYYGMDV 72.0 7D
5-10-1*01_3-10*01_3*02_M_JF274048 ARRATYYYGSGSYFDAFDI Anti-Influenza H5N1 Viruses
4-30-4*01_3-0*01_3*02_UM_GU358680 AR-KTYYYGSGSYYDAFDI 89.7
1-69*13_2-2*01_6*02_UM_U86795 AGT-IVVVPAAGGIFFYYYGMDV Heterohybridoma derived from CD5+ CLL B lym-
phocytes with rheumatoid factor activity
1-69*01_2-2*01_6*02_UM_HM173323 ARTDIVVVPAAMII-YYYYGMDV 72.7
1-69*06_3-16*02_3*02_UM_AF460965 ARGGNYDYIWGSYRSNDAFDI Antiphospholipid antibodies
1-69*01_3-16*02_3*02_UM_EF091909 ARGGDYDYIWGSYRPNDAFDI 90.5 6
1-69*01_3-16*02_3*02_UM_GU358676 ARGGNYDYIWGSYRTNDAFDI 95.0 6
PH0955 ARVSIFGVVQHYYYYYYMDV Rheumatoid factor
1-69*01_3-3*01_6*02_UM_KC802106 ARVQVFGVVNTYYYYYYMDV 80.0
TT genotype
4-34*01_3-3*01_6*03_UM_LM647851 ARGRVGYDFWSGS-PYYYYYYMDV Peripheral blood B cell
4-34*01_3-3*01_6*03_UM_EF091922 ARGFGYYDFWSGTHPPNYYYYMDV 70.8
3-21*01_6-13*01_6*02_UM_LM647465 ARDQGSSSSWFDYYYYGMDV Peripheral blood B cell
3-21*01_6-13*01_6*02_UM_EF091900 ARDRGVSSSWYLSYYYYMDV 70.0 26
1-8*01_6-13*01_4*01_UM_LM647894 AR-GARYSSSWYPFDY Peripheral blood B cell
3-30*01_6-13*01_4*01_UM_HM173331 ARVGTGYSSSWYPFDY 81.0
3-74*02_3-3*01_6*02_UM_LM647144 AAIYDFWSGYWSYYYYYGMDV Peripheral blood B cell
3-48*03_3-3*01_6*02_UM_JQ928950 ARDYDFWSGYYAYYYYYGMDV 31 80.9
3-7*01_3-3*01_6*02_UM_LM648392 ARGLYDFWSGYYPHYYYYGMDV Peripheral blood B cell
1-69*01_3-3*01_6*02_UM_S17 AAQ--DFWSGYYPHYYYYGMDV 60.0
3-64*01_2-2*03_6*02_UM_EF541666 ARDGYCSSTSCYLDGGLYYYY-GMDV Ig from X-HIgM patient
1-2*04_2-15*01_6*02_UM_JF810257 AREGYCSGGSCYPPPGNYYYYYGMDV 69.0 UA/ITA_17
3-48*02_3-3*01_6*02_UM_AF077457 ARDSTIFGVVII-DYYYGMDV Ig from X-HIgM patient
3-30*03_3-3*01_6*02_UM_EU433876 RAQM-IFGVVIIEDYYYGMDV 70.0 9
3-30*03_-3*01_6*02_UM_EF542369 ASHYDFWSGHYEPYYYYGMDV Ig from X-HIgM patient
3-48*03_3-3*01_6*02_UM_JQ928950 ARDYDFWSGYYAYYYYYGMDV 31 80.9
1-2*04_3_3*01_6*02_UM_AY686915 ARES--YYDFWSGKRN-YYYYGMDV Anti-rotavirus Ig
1-2*04_3_3*01_6*02_UM_EU433869 ARDGLQYYDFWSGSDLAYYYYGMDV 68.0 7D
3_11*05_3-3*01_5*02_UM_DQ322854 ARDTRPYDFWSGYYP-NWFDP Anti-pneumococcal polysaccharides Ig
4-61*01_3-3*01_5*02_UM_EF091926 ARHRGDYDFWSGYYPYNWFDP 80.9 UA_5
4-59*01_3-3*01_6*03_UM_FJ385336 ARDLTYYDFWSGYYPDYYYY-MDV Anti-respiratory syncytial virus Ig
4-34*01_3-3*01_6*03_UM_EF091922 ARGFGYYDFWSGTHPPNYYYYMDV 66.6
1-2*02_1-26*01_6*02_UM_AY944713 ARVYSGSYPYYYYGMDV Anti-rotavirus Ig
1-2*02_1-26*01_6*02_UM_EF091912 ARPYSGSYPWYYYGMDV 88.0 28
3-21*01_6-13*01_6*02_UM_KJ409179 ARDKEYSSSWYLYPYYYYMDV Anti-HCV Ig
3-21*01_6-13*01_6*02_UM_EF091900 ARDRGVSSSWYL-SYYYYMDV 75.0 26
1-18*01_1-26*01_4*02_M_JX213637 ARDVQYSGSYLGAYYFDY Anti-influenza B viruses Ig
4-59*01_1-26*01_4*02_UM_JX462751 ARHDPYSGSYLV-YYFDY 70.5
3-11*01_3-3*01_5*02_UM_DQ322847 ARDTRPYDFWSGYYP-NWFDP Anti-pneumococcal polysaccharides Ig
3-11*01_3-3*01_5*02_UM_EU667604 ARD-R-YDFWSGYIGYNWFDP 73.6 UA_5
4-34*01_3-3*01_4*02_UM_X54441 ARGGSVLRFLEWLLYPAFDY Rheumatoid factor
1-69*01_3-3*01_4*02_UM_EU667599 ARSGE-LRFLEWLLSADFDY 68.4 UA_7
3-33*01_4-17*01_3*02_UM_AJ305181 AKGDYGDYSFAFDI Anti-SLE Ig
3-33*01_4-17*01_3*02_UM_EF407846 ARGVPGDYVMAFDI 71.0 UA_9
3-30-3*01_4-17*01_3*02_UM_EF407832 ARGPRGDYVSSFDL 57.0 UA_9
��6 Experimental Oncology ��� �������� ���� ��eptem�er�
mutated �mutations within exon 3� and non-coding
region�. The occurrence of UM IGHV genes of I clan
�except IGHV1-69) among NOTCH1-mutated cases
was twice as high as in the general group. Therefore�
it can �e suggested that expression of UM IGHV genes
of I clan �except IGHV1-69) is a risk factor for the
presence of NOTCH1 mutations. On the contrary� the
pro�a�ility of detecting NOTCH1 mutations was lower
in the carriers of other UM genes �it should �e noted
that su�set #� was a�sent in our cohort�.
When searching for non-coding mutations
in the 3´UTR region of the NOTCH1� we paid attention
to rs3���59�� which is localized in the amplified region.
Preliminary data suggested its functional signifi-
cance. Quan et al. found association of CC genotype
of rs3���59� ��6.�% among cases and ��.�% in the
control group� with the risk of lung cancer in north-
east Chinese non-smoking females [��]. In study
of Gao et al.� also performed in Chinese population�
CC genotype of rs3���59� was a�sent in the control
group �n = ���� and among patients with invasive
ductal carcinoma �IDC� n = ���� and ductal carcinoma
in situ �DCI�� n = 5�� [�5]. The C allele was significantly
associated with high risk of DCI�� �ut not IDC; the
TC genotype was significantly associated with an in-
creased risk of IDC and DCI� and poorly differentiated
IDC. In addition� Notch� protein expression was signifi-
cantly higher in DCI� patients with the TC genotype.
Although Notch� protein expression was higher in IDC
patients with the TC genotype� this association did not
reach significance. Authors concluded that the impact
of the rs3���59� variant on Notch� protein expression
mainly occurs early in IDC development.
In our study� rs3���59� did not affect the risk of CLL
and survival parameters of patients. At the same time�
differences were found in the frequency of IGHV gene
usage and in the structure of HCDR3 in carriers of indi-
vidual genotypes. Leukemic cells of CC homozygotes
expressed the most limited spectrum of UM IGHV
genes �mostly IGHV1-69 and IGHV3-11�� and their
HCDR3 sequences were homologous only with the
sequences of normal B-cells. Conversely� leukemic
cells of TT homozygotes used the largest num�er
of UM IGHV genes� more frequently had HCDR3 ho-
mology with anti�acterial or antiviral Ig clones� had
less N nucleotide additions in DJH junctions� and the
num�er of sequences that lacked N additions at DJH
junctions had a tendency to �e higher. By two last
parameters� they were a �it like a memory B cells�
characterized �y Tian et al. [��]. It is known that
Notch� signaling regulates B and T lymphocyte de-
velopment [���3�]. Taking into account data of Cao
et al. [�5] on the functional significance of rs3���59��
we hypothesized that rs3���59� could influence on the
selection of B-cell clones during early stages of CLL
development. This assumption are in an agreement
with reports regarding the role and possi�le mecha-
nisms of NOTCH signaling in regulation of the normal
B-cell repertoire� summarized �y Cruickshank and
Ulgiati [3�].
ACKNOWLEDGMENT
The authors are grateful to Mr. Thomas Harms�
President of Charita�le Organization “KIHEW-Kinder-
hilfe” �Germany� who provided support �y reagents
for fulfillment of the work.
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