Pathological

Communication

Biosci. Biotech. Res. Comm. 10(3): 391-397 (2017)

Comparison of multiplex ligation-dependent probe

ampli cation and qPCR for screening PAX5 gene

detection in acute lymphoblastic leukemia

Sahar Mehranfar

1, 2

, Sirous Zeniali

3,4

, Nasser Samadi

, Nazanin Maleki Sadeghi

and

Abbasali Hosein Pour Feizi

1,7

Hematology & Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Department of Molecular Medicine, School of Advanced Medical Science, Tabriz University of Medical

Science, Tabriz, Iran

Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Kawsar Human Genetics Research Center, Kawsar Genomics Center, Tehran, Iran

Department of Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

Lorestan University of Medical Sciences, Khorramabad, Iran

ABSTRACT

Despite the advances in diagnosis of leukemia, still we need a rapid and cost-bene t screening method in patients

with acute lymphoblastic leukemia (ALL). The aim of the present study is to evaluate the ability of Multiplex Ligation-

dependent Probe Ampli cation (MLPA) method in screening patients with ALL. 45 patients with ALL were selected. DNA

were extracted, then PAX5 gene copy number abnormalities (CNAs) were studied by adopting MLPA and QPCR methods.

To prove the results of two methods, PCR product from three samples were sent for sequencing. From 45 patients with

B-ALL, 11 (24%) patients, showed CNAs after applying MLPA method. From among all mutations, 22 samples (29%)

were seen in PAX5 gene. We used Sanger sequencing as a gold standard method to compare the two methods. After

sequencing, we were submitted 2 genes in Gene Bank by accession numbers (ID) KX608846 and KX707789 in exon 10

of PAX5. MLPA is a rapid and valid method for screening of genes mutation and can be used in these laboratories as

routine method especially in low-income countries.

KEY WORDS: ACUTE LYMPHOBLASTIC LEUKEMIA, COPY NUMBER ABNORMALITY, MLPA, QPCR

391

ARTICLE INFORMATION:

*Corresponding Author: pourfeizi@yahoo.com

Received 2

July, 2017

Accepted after revision 22

Sep, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/10.3/9

Sahar Mehranfar et al.

INTRODUCTION

Today, B-cell acute lymphoblastic leukemia (B-ALL) is the

most common childhood malignancy, especially in chil-

dren below  ve years of age. Today, we have extensive

knowledge about leukemia like other cancers. In the last

two decades, molecular methods have provided the physi-

cians and researchers with a new vision towards the role

of molecular factors in pathogenesis of diseases. Alterna-

tive expression leads to the abnormal proliferation and

differentiation of lymphoid ancestors (Fazio, Biondi, &

Cazzaniga, 2011; O’neil & Look, 2007 Seiter etal., 2014).

Paired Box5 (PAX5) is an important transcription

factor located on chromosome 9p13 and includes 10

exons. PAX5 is essential factor for the development

of pro-B cell to mature B-cell(Fazio etal., 2011). Up to

now, nine PAX transcription factors have been demon-

strated, although, PAX5 is the only PAX protein which is

expressed in the hematopoietic system (Busslinger, 2004;

Cobaleda, Schebesta, Delogu, & Busslinger, 2007; Mat-

thias & Rolink, 2005). The improvement of diagnostic

and treatment protocols by using risk-adapted methods

have enhanced cure rate up to 80%(Carter etal., 2001).

Therefore, the identi cation of biomarkers can be a

great help for the patients and the health-care system to

reduce the health-care costs and side effects of chemo-

therapy. Screening is highly pivotal in the developing

countries where the  nancial resources and drugs are

limited. Childhood cancer rates has been rising slightly

in the past few decades (Fathi etal., 2015).

In Iran, leukemia has an increasing trend and leaves

a heavy burden on the whole society,(Fazeli etal., 2013).

Thus, more attention needs to be paid to screening, early

diagnosis, and effective treatment in order to increase the

survival rate for children’s cancer (Mousavi etal., 2008).

In this study, we carried out Multiplex Ligation-depend-

ent Probe Ampli cation (MLPA) analysis on 45 ALL patients’

samples to determine the copy number abnormality (CNA)

of PAX5 gene. MLPA is a technique capable of showing

variations in the copy number of several human genes.Due

to this capability, MLPA is mostly used in the molecular

diagnosis of several genetic diseases whose pathogenesis

is related to the presence of deletions or duplications of

speci c genes,(Schouten etal., 2002; Stuppia, Antonucci,

Palka, & Gatta, 2012). Also, we adopted the real-time PCR

method for comparing the results of two methods.

MATERIALS AND METHODS

58 Bone marrow(BM) or/and peripheral blood (PB) sam-

ples were taken at the time of diagnosis of patients with

B-ALL, who were presented to the Children’s Hospital

of Tabriz University of Medical Sciences, between 2014

and 2016. The selection criteria included having at least

20% blast cells, and being between 1-14 years of age

at the time of diagnosis. The exclusion criteria were the

samples from foreign patients and DNA with insuf -

cient quality. Six samples were excluded due to failure

of MLPA, Five samples due to bad quality of DNA and

two of them were withdrawn. Additionally, 4 bone mar-

row and 7 peripheral blood samples from healthy donors

were analyzed as control samples. Graphical abstract of

diagnosis process in this study are shown in (Figure 1).

The written informed consent was obtained from all

the parents and the study protocol was approved by Eth-

ics and Human Rights Committee of Tabriz University of

Medical Sciences.

The DNA was extracted from BM or PB samples,

using the QIAamp DNA minikit (Qiagen, Hilden, Ger-

many) according to the manufacturer’s

instructions. In

order to verify the  delity of DNA concentration and to

evaluate its quality, a spectrophotometer,(NanoDrop,Wi

lmingtonDE,,USA) and 1.5% agarose electrophoresis gel

were used respectively.

Two pairs of primer for exone10 of PAX5 gene were

designed using the online program oligo7. The primer

sequences are shown in (Table 1). SYBR Green Real-time

PCR (BIO-RAD iQ5, Bio-Rad, Hercules, California, USA)

was done using a serial dilution of DNA samples includ-

ing 100, 50, 25, 12.5 and 6.25ng in triplicate repeats.

Then, according to the standard curves and by compar-

ing the slope and ef ciency of each reaction (Schmitt-

gen, Lee, & Jiang, 2008), 50 ng of DNA were preferable

as the best concentration. The analysis was performed

in a total volume of 20 l including 50 ng of DNA, 0.2

mM speci c primers and 10 l of SYBR Green Master

mix (Applied Biosystems, Foster City, California, United

States) according to the manufacturer’s instructions,

then (2

−∆∆Ct

) was calculated by normalizing samples and

the relative gene copy numbers were calculated. The

FIGURE 1. Graphical

abstract of diagnostic

process.

392 COMPARISON OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION AND QPCR BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARISON OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION AND QPCR 393

Sahar Mehranfar et al.

copy numbers compared to the reference gene were

determined. Following values were considered for result

analysis: 1 for normal samples, 0.5 for heterozygous

deletions, and 1.5 for heterozygous duplications. Results

were analyzed using Microsoft Excel.

The MLPA probe sizes, chromosomal position and

sequences and other complete details are available on the

Website (www.MRC-Holland.com). The sensitivity of the

MLPA kit P335-B2 was assessed previously by Kuiperetal

(Kuiper etal., 2007) . In each run of MLPA, appropriate

numbers of the samples, and three to six control samples

were used in same run. All control samples were taken

from the children with no acute leukemia or other types

of malignancy. In this study, 50 ng of genomic DNA in

 nal volume of 5 µl were used to determine copy number

of PAX5 gene by using the SALSA MLPA kit P335-B2

(MRC Holland, The Netherlands). Capillary electropho-

resis were run on 3130XL Genetic Analyzer (Life Tech-

nologies, Carlsbad, CA, USA) and ampli ed PCR products

were analyzed by Gene Marker version 1.95 (Soft Genet-

ics State College, Pennsylvania, USA). Values over < 0.75

and > 1.35 were considered to be abnormal and values

below 0.25 indicated biallelic deletion. Schematic MLPA

analysis is shown in (Figure 2).

Normality of data was calculated by Kolmogorov-

Smirnov test and was presented by Mean and Stand-

ard Deviation. Non-normal variables were incidental as

Median (Max & Min). The gene CNAs difference between

the ALL and control samples were calculated by inde-

pendent sample T-test and regression tree for accuracy

of MLPA method. P-values less than 0.05 were consid-

ered statistically signi cant. All statistical analyses were

done using the Statistical package for the Social Sci-

ences (SPSS), version 16.0 (SPSS Inc, Chicago, IL, USA).

RESULTS AND DISCUSSION

45 patients with ALL, including 27 (60%) boys with

mean age of 5.8±3.4 and 18 (40%) girl with mean age

of 6.0 ±2.7 years were studied (p>0.05). Statistically, no

signi cant differences were seen between the mean ages

of genders. Demographic data of the participants have

been summarized in (Table 2).

We found a wide range of alterations in the PAX5

gene in this study; deletion or duplication in one or

more exons. In 2 cases, deletion and duplication were

seen in different exons simultaneously. From among all

patients, 11 (24%) patients showed 76 CNAs, from which,

22 (29%) were seen in PAX5 gene. The more frequent

exons were 6, 7, and 8 (55%); the least one was exon

1 (4%). No signi cant difference was observed between

CNAs and sex, age, National Cancer Institute (NCI) risk

group or source of samples (p<0.05). Schematic detected

CNAs are shown in (Figure 3).

From among all samples, three samples showed

changes in exon 10 by MLPA. All of these samples (3)

were validated by real-time PCR. The standard melting

curve of real-time PCR is shown in (Figure 1).

The accuracy obtained for MLPA method for detec-

tion of CNAs comparisons of real-time PCR was 86%.

To prove the results of two methods, PCR product from

these patients was sent to Bioneer for sequencing. The

sequencing analyses were done by Chromas software,

and the results were compared with the reported gene

sequences. We submitted 2 genes in GenBank by acces-

sion numbers (ID) KX608846 and KX707789 in exon 10

of PAX5. Schematic representation of each method is

shown in (Figure 4).

ALL is a heterogeneous disease with differing in

response to chemotherapy. The results of some studies

demonstrated that the identi cation of molecular mark-

ers may improve the treatment approaches (Yang etal.,

2011). Today, the determination of gene dosage is impor-

tant for both clinical and research medicine and it is also

required for therapeutic, prognostic, or diagnostic goals

(Ginsburg & Willard, 2009; Vogelstein & Kinzler, 2004).

Due to the growing need for molecular studies, a lot of

new molecular markers have been discovered (Mrózek,

Table 1. The primer sequencing used in QPCR for PAX5 copy number abnormalities.

Gene name Product length Tm(Cº) Primer sequence

PAX5 F1 170 64 5-ATCTTAACCTAGGCAGAGCATC-3

PAX5 R1 170 61 5-CAAGAGACACACCATTTGGG-3

PAX5 F2 164 62 5-CTCCTTCTTAGTATCTACGAG-3

PAX5 R2 164 60 5-GAACTCAAAGAAACTGTCTGG-3

GAPDH F 120 60 5 -CGGTGGATCCCTTTAATTG-3-3

GAPDH R 120 58 5 –CAATAAAGGGGATCCACCG-3

Sahar Mehranfar et al.

Döhner, & Bloom eld, 2007; Thiede et al., 2006). It

seems that in the future, patients could be divided into

two groups in terms of presence or absence of these bio-

markers (Mi etal., 2012).

PAX5 translocations do not in uence the progno-

sis or outcome by themselves but it is in association

with other molecular aberrancies in theirs pathway

that they in uence the prognosis or outcome (Sellner &

Taylor, 2004). Similar to our  ndings, the incidence of

PAX5rearrangements was seen about 30% in the B-cell

precursor ALL, in a previous study (Barbosa etal., 2015;

Hunger & Mullighan, 2015; C. Schwab etal., 2010; C. J.

Schwab etal., 2013). Array-based approaches can pro-

vide high resolution data on CNAs, but these methods

are restricted by low throughput, high cost, and time

consumption. Therefore, the need for a simple, fast and

cost-ef cient method to screen molecular changes has

remained hitherto. (Vermeesch etal., 2007).

The PCR-based multiplex ligation-dependent probe

ampli cation (MLPA) technique is a relative quanti-

 cation method for gene dosage determination and

deletions/duplications mutations in unknown samples

which are recognized by comparison to the normal and

standard control (Al Zaabi, Fernandez, Sadek, Riddell,

& Greer, 2010). Buijs etal. performed genomic pro ling

using MLPA in 45 cases with (ALL), showing that MLPA

is able to detect anomalies similar to PCR method (Buijs,

Krijtenburg, & Meijer, 2006).

A similar study was done by Coll-Mulet etal., who car-

ried out MLPA in 50 chronic leukemia patients. Their results

illustrated that only cases with a low percentage (<25%) of

cells, were not detected by MLPA (CollMulet etal., 2008).

Many other studies on different types of leukemia revealed

excellent accuracy and speci city of MLPA as compared to

QPCR and showed clinical capability of these techniques

with different disease(Abdool, Donahue, Wohlgemuth, &

Yeh, 2010; Donahue, Abdool, Gaur, Wohlgemuth, & Yeh,

2011; Fabris etal., 2011; Mullighan etal., 2008; C. Schwab

etal., 2010; Schwartz & Dunø, 2004).

However, there are still some limitations such as being

unable to detect balanced rearrangements, mosaicism,

heterogeneity or contamination with normal cells. Like

PCR, MLPA reactions are also more sensitive to contami-

nants (Stuppia etal., 2012; Tavtigian & Le Calvez-Kelm,

2007). According to the manufacturer’s instructions of

MLPA, it works with only 20 ng of DNA. Besides, the

results don’t depend on the amount of used sample DNA

but, our study showed that 50 ng are required for reli-

able results.

Moreover, it is recommended to compare differ-

ent MLPA analyses only by using one DNA extraction

method from different sources of samples from each

patient, to compare specify the sensitivity of this method

for different sources of samples. Real-time PCR method

is highly sensitive and speci c as reported in many stud-

ies all over the world, though, it is a time-consuming

method and can survey only one target sequence in per

run (Ponchel etal., 2003; Ramalingam etal., 2009).

But Compared to QPCR, MLPA is a low cost, fast, and

technically uncomplicated method for the analysis of

FIGURE 2. MLPA analysis sheet; a: compares peaks between

standard and patients probes; b: peaks ratio; c: demonstrated

probes ratio in cut-off range (Red peak: mean of whole sam-

ples peak, Blue peaks: samples peak, Black peak: reference

peak).

394 COMPARISON OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION AND QPCR BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARISON OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION AND QPCR 395

Sahar Mehranfar et al.

Table 2. Demographic data of acute lymphoblastic

leukemia patients

Variable Frequency (Percent)

Sex

Female 18(40%)

Male 27(60%)

Age

1-4 22(48%)

5-10 17(37%)

10-14 6(13%)

Mean of age

Female 6.0 ±2.7

Male 5.8±3.4

NCI risk group

Standard risk 28(62%)

High risk 17(37%)

Source of

samples

BM samples 19(42%)

PB samples 26(57%)

WBC count

>50,000 11

<50,000 34

FIGURE 3. Schematic representation of the CNAs found in this study. PAX5 exons (1-10) are shown

above. Numbers in the red shapes show the occurrence times for each rearrangement.

results. Moreover, in the future, MLPA could be applied

to large CNAs screening. PCR and sequencing can be

used for con rming mutation only in selected gene seg-

ments, but MLPA can be considered for screening a large

area of genes, simultaneously.

CONCLUSION

Molecular screening is an essential test for comprehen-

sive survey of disease, and provides complementary

information for better diagnosis, treatment and follow-

up of patients with acute leukemia. This approach may

be useful as a criterion for measuring the effectiveness

of new molecular tools such as genetic proﬁling. Con-

sidering differences between two methods, more clini-

cal studies are required to  nd out the best one for our

objectives. Our data clearly indicated that MLPA can

be an attractive alternative method to other molecular

and cytogenetic techniques that are now routinely used.

Screening of critical genes in pathobiology of all cancers

such as acute lymphoblastic leukemia could help early

diagnosis in the early stages. It is especially useful when

the inexpensive and available techniques are used eve-

rywhere. It has been established that early diagnosis has

a positive impact on the prognosis.

Sahar Mehranfar et al.

REFERENCES

Abdool, A ., Donahue, A. C., Wohlgemuth, J. G., & Yeh, C.-H.

(2010). Detection, analysis and clinical validation of chro-

mosomal aberrations by multiplex ligation-dependent probe

ampli cation in chronic leukemia. PLoS One, 5(10), e15407.

Al Zaabi, E. A., Fernandez, L. A., Sadek, I. A., Riddell, D. C., &

Greer, W. L. (2010). Multiplex ligation-dependent probe ampli-

 cation versus multiprobe  uorescence in situ hybridization to

detect genomic aberrations in chronic lymphocytic leukemia: a

tertiary center experience. The Journal of molecular diagnos-

tics, 12(2), 197-203.

Barbosa, T. C., Terra-Granado, E., Magalhães, I. M. Q., Neves,

G. R., Gadelha, A., Guedes Filho, G. E., . . . Pombo-de-Oliveira,

M. S. (2015). Frequency of copy number abnormalities in com-

mon genes associated with B-cell precursor acute lymphoblas-

tic leukemia cytogenetic subtypes in Brazilian children. Cancer

genetics, 208(10), 492-501.

Buijs, A., Krijtenburg, P. J., & Meijer, E. (2006). Detection of risk-

identifying chromosomal abnormalities and genomic pro ling

by multiplex ligation-dependent probe ampli cation in chronic

lymphocytic leukemia. Haematologica, 91(10), 1434-1435.

Busslinger, M. (2004). Transcriptional control of early B cell

development 1. Annu. Rev. Immunol., 22, 55-79.

Carter, T. L., Watt, P. M., Kumar, R., Burton, P. R., Reaman, G.

H., Sather, H. N., Kees, U. R. (2001). Hemizygous p16 INK4A

deletion in pediatric acute lymphoblastic leukemia predicts

independent risk of relapse. Blood, 97(2), 572-574.

FIGURE 4. The comparison of the results of 3 methods; a: sequenc-

ing result, b: QPCR result, c: MLPA result.

Cobaleda, C., Schebesta, A., Delogu, A., & Busslinger, M.

(2007). Pax5: the guardian of B cell identity and function.

Nature immunology, 8(5), 463-470.

CollMulet, L., Santidrián, A. F., Cosialls, A. M., IglesiasSerret,

D., De Frias, M., Grau, J., Domingo, A. (2008). Multiplex liga-

tiondependent probe ampli cation for detection of genomic

alterations in chronic lymphocytic leukaemia. British journal

of haematology, 142(5), 793-801.

Dona hue, A. C., Abdool, A. K., Gaur, R., Wohlgemuth, J. G., &

Yeh, C.-H. (2011). Multiplex ligation-dependent probe ampli -

cation for detection of chromosomal abnormalities in myelo-

dysplastic syndrome and acute myeloid leukemia. Leukemia

research, 35(11), 1477-1483.

Fabr is, S., Scarciolla, O., Morabito, F., Cifarelli, R. A., Dininno,

C., Cutrona, G., . . . Ciceri, G. (2011). Multiplex ligationdepend-

ent probe ampli cation and  uorescence in situ hybridization

to detect chromosomal abnormalities in Chronic lymphocytic

leukemia: A comparative study. Genes, Chromosomes and

Cancer, 50(9), 726-734.

Fathi , A., Amani, F., & Bahadoram, M. (2015). Epidemiology of

Childhood Cancer in Northwest Iran. Asian Paci c Journal of

Cancer Prevention, 16(13), 5459-5462.

Fazel i, Z., Pourhoseingholi, M. A., Vahedi, M., Abadi, A.,

Bavand-Pour, F. S. F., & Baghestani, A. R. (2013). Leukemia

cancer mortality trend in Iran, from 1995 to 2004. Iranian

journal of cancer prevention, 6(3), 170.

Fazio , G., Biondi, A., & Cazzaniga, G. (2011). The Role of PAX5

in ALL: Citeseer.

396 COMPARISON OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION AND QPCR BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARISON OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION AND QPCR 397

Sahar Mehranfar et al.

Ginsb urg, G. S., & Willard, H. F. (2009). Genomic and person-

alized medicine: foundations and applications. Translational

research, 154(6), 277-287.

Hunge r, S. P., & Mullighan, C. G. (2015). Acute lymphoblas-

tic leukemia in children. New England Journal of Medicine,

373(16), 1541-1552.

Kuipe r, R., Schoenmakers, E., Van Reijmersdal, S., Hehir-Kwa,

J., van Kessel, A. G., Van Leeuwen, F., & Hoogerbrugge, P.

(2007). High-resolution genomic pro ling of childhood ALL

reveals novel recurrent genetic lesions affecting pathways

involved in lymphocyte differentiation and cell cycle progres-

sion. Leukemia, 21(6), 1258-1266.

Matth ias, P., & Rolink, A. G. (2005). Transcriptional networks

in developing and mature B cells. Nature Reviews Immunol-

ogy, 5(6), 497-508.

Mi, J ., Wang, X., Yao, Y., Lu, H., Jiang, X., Zhou, J. Tang,

J. (2012). Newly diagnosed acute lymphoblastic leukemia in

China (II): prognosis related to genetic abnormalities in a series

of 1091 cases. Leukemia, 26(7), 1507-1516.

Mousa vi, S., Alamolhoda, A., Gouya, M., & Lickiss, N. (2008).

Implementation of Comprehensive National Cancer Con-

trol Program in Iran: an experience in a developing country.

Annals of Oncology, 19(2), 398-400.

Mróze k, K., Döhner, H., & Bloom eld, C. D. (2007). In uence of

new molecular prognostic markers in patients with karyotypi-

cally normal acute myeloid leukemia: recent advances. Current

opinion in hematology, 14(2), 106-114.

Mulligh an, C. G., Miller, C. B., Radtke, I., Phillips, L. A., Dal-

ton, J., Ma, J., . . . Pui, C.-H. (2008). BCR–ABL1 lymphoblastic

leukaemia is characterized by the deletion of Ikaros. Nature,

453(7191), 110-114.

O’neil, J., & Look, A. (2007). Mechanisms of transcription fac-

tor deregulation in lymphoid cell transformation. Oncogene,

26(47), 6838-6849.

Ponchel , F., Toomes, C., Brans eld, K., Leong, F. T., Douglas,

S. H., Field, S. L. Mighell, A. J. (2003). Real-time PCR based

on SYBR-Green I  uorescence: an alternative to the TaqMan

assay for a relative quanti cation of gene rearrangements,

gene ampli cations and micro gene deletions. BMC biotech-

nology, 3(1), 1.

Ramalin gam, N., Liu, H.-B., Dai, C.-C., Jiang, Y., Wang, H.,

Wang, Q. Gong, H.-Q. (2009). Real-time PCR array chip with

capillary-driven sample loading and reactor sealing for point-

of-care applications. Biomedical microdevices, 11(5), 1007-

1020.

Schmitt gen, T. D., Lee, E. J., & Jiang, J. (2008). High-through-

put real-time PCR. Molecular Beacons: Signalling Nucleic Acid

Probes, Methods, and Protocols, 89-98.

Schoute n, J. P., McElgunn, C. J., Waaijer, R., Zwijnenburg, D.,

Diepvens, F., & Pals, G. (2002). Relative quanti cation of 40

nucleic acid sequences by multiplex ligation-dependent probe

ampli cation. Nucleic acids research, 30(12), e57-e57.

Schwab, C., Jones, L., Morrison, H., Ryan, S., Yigittop, H.,

Schouten, J., & Harrison, C. (2010). Evaluation of multiplex

ligationdependent probe ampli cation as a method for the

detection of copy number abnormalities in Bcell precursor

acute lymphoblastic leukemia. Genes, Chromosomes and Can-

cer, 49(12), 1104-1113.

Schwab, C . J., Chilton, L., Morrison, H., Jones, L., Al-Shehhi,

H., Erhorn, A., . . . Harrison, C. J. (2013). Genes commonly

deleted in childhood B-cell precursor acute lymphoblastic

leukemia: association with cytogenetics and clinical features.

Haematologica, haematol. 2013.085175.

Schwartz, M., & Dunø, M. (2004). Improved molecular diagno-

sis of dystrophin gene mutations using the multiplex ligation-

dependent probe ampli cation method. Genetic testing, 8(4),

361-367.

Seiter, K. , Sarkodee-Adoo, C., Talavera, F., Sacher, R., & Besa,

E. (2014). Acute lymphoblastic leukemia. Sarkodee-Adoo

C, Talavera F, Sacher RA and Besa EC: Medscape Reference.

WebMD. Accessed, 17.

Sellner, L . N., & Taylor, G. R. (2004). MLPA and MAPH: new

techniques for detection of gene deletions. Human mutation,

23(5), 413-419.

Stuppia, L ., Antonucci, I., Palka, G., & Gatta, V. (2012). Use

of the MLPA assay in the molecular diagnosis of gene copy

number alterations in human genetic diseases. International

journal of molecular sciences, 13(3), 3245-3276.

Tavtigian, S. V., & Le Calvez-Kelm, F. (2007). Molecular Diag-

nostics: Methods and Limitations. Hereditary Breast Cancer,

179-205.

Thiede, C. , Koch, S., Creutzig, E., Steudel, C., Illmer, T., Schaich,

M., & Ehninger, G. (2006). Prevalence and prognostic impact

of NPM1 mutations in 1485 adult patients with acute myeloid

leukemia (AML). Blood, 107(10), 4011-4020.

Vermeesch, J. R., Fiegler, H., de Leeuw, N., Szuhai, K., Schou-

mans, J., Ciccone, R. Van Ravenswaaij, C. (2007). Guidelines

for molecular karyotyping in constitutional genetic diagnosis.

European Journal of Human Genetics, 15(11), 1105-1114.

Vogelstein , B., & Kinzler, K. W. (2004). Cancer genes and the

pathways they control. Nature medicine, 10(8), 789-799.

Yang, Y. L ., Hung, C. C., Chen, J. S., Lin, K. H., Jou, S. T., Hsiao,

C. C.Lin, S. R. (2011). IKZF1 deletions predict a poor prognosis

in children with Bcell progenitor acute lymphoblastic leuke-

mia: A multicenter analysis in Taiwan. Cancer science, 102(10),

1874-1881.