Medical Research Updates ⌠MRU⌡. ORIGINAL RESEARCH ARTICLE

MRU 2024; Vol.1(Issue2): 19-24 ISSN 200XX |

DOI: https://www.mruj.online/_downloads/c4d0aa9d3b9526980434be67dff8a1a8

MLH1 and MSH2 gene mutation patterns in Lynch syndrome-associated colorectal

cancer in Sudan

Balgis Elhag Ibrahim Tager1, Salah Eldin G. Elzaki2, and Ahmed Abdula Agabeldour 3

1Department of Histopathology and Cytology, Faculty of Medical Laboratory Sciences, West

Kordofan University, Elnuhood, Sudan.

2Department of molecular epidemiology, Tropical Medicine Research Institute, National

Centre for Research Khartoum Sudan.

3Department of Pathology, Faculty of Medicine, Kordofan University, El-Obeid, Sudan.

ABSTRACT

Background: Colorectal cancer (CRC) is increasingly becoming dominant in Sudan, which is attributed to several

factors, including hereditary mutations in the DNA mismatch repair genes MLH1 and MSH2. Therefore, this study

aimed to examine the MLH1 and MSH2 gene mutation patterns in Lynch syndrome-associated colorectal cancer

in a series of Sudanese patients with CRC. Methodology: This study investigated 50 patients with CRC who

attended El-Obeid Hospital during the period from 2017 to 2022. The presence of MLH1 and MSH2 was indicated

by immunohistochemical testing of formalin-fixed, paraffin-wax-embedded tissues. Results: The MLH1 mutation

was positive in 28% of the study population and negative in 72% of them. The MSH2 mutation was positive in

42% and negative in 58% of the study subjects. The MLH1 and MSH2 mutations shared positivity in 18% and

negativity in 24% of the cases. Conclusion: According to the present studies, CRC is more prevalent in women. LS

is more common in Sudanese patients with CRC than in many other reports throughout the world. MSH2

mutations are more prevalent in Sudanese patients than MLH1 mutations.

Keywords: colorectal cancer, Lynch syndrome, MLH1, MSH2, Sudan. ..

Correspondence to: Balgis Elhag Ibrahim Tager, Email: bintelhag@yahoo.com

Cite this article: Tager BEI, Elzaki SGE2, and Agabeldour AA. Medical Research Updates 2024;1(2): 1-5. DOI:

https://www.mruj.online/_downloads/c4d0aa9d3b9526980434be67dff8a1a8

INTRODUCTION

Lynch syndrome (LS), formerly known as

hereditary non-polyposis colorectal cancer

(HNPCC), is an autosomal dominant illness

characterized by mutations in the DNA mismatch

repair genes MLH1, MSH2, MSH6, and PMS2. LS

predisposes to a wide range of malignancies, the

most common of which are colorectal tumors. The

key risk factors for LS include gender, age, and the

genes implicated [1]. Autoimmunity and

immunodeficiency aid in the development of

malignancies in LS [2]. More than 90% of CRC cases

are adenocarcinomas in glandular epithelial cells of

the large intestine (colon and rectum), with the

remaining 5% being hereditary nonpolyposis CRC

(HNPCC) or familial adenomatous polyposis (FAP)

caused by APC, MLH1, and MSH2 mutations [3, 4].

DNA mismatch repair (MMR) gene homozygous

and heterozygous germline mutations are the

cause of Lynch syndrome. MSH2 accounts for about

40–50%, MLH1 (30–37%), and MSH6 mutations

are detected in 7–13% and up to 9 percent of PMS2

cases. Patients with LS have a lifetime chance of

acquiring colorectal cancer of 50–80% [5].

Mismatch repair (MMR) proteins (MLH1, PMS2,

MSH2, and MSH6) are the major DNA repair system

that repair mismatches and small insertions and

deletions that occur during cellular replication;

their deficiency (MMR-D) affects microsatellites

and causes alterations known as microsatellite

instability (MSI), as well as loss of MMR protein

expression in tumors. CRC is generally sporadic,

with approximately 5% to 10% being hereditary

colon cancer syndromes, which include Lynch

syndrome, adenomatous polyposis syndromes, and

hamartomatous polyposis syndromes. [6].

Colorectal cancer (CRC) is one of the most frequent

malignancies globally; approximately 20–30% of

CRCs are familial, and LS is the most common form

of hereditary CRC caused by germline mismatch

repair (MMR) gene mutations (MLH1, MSH2,

MSH6, or PMS2) [5]. However, there is a scarcity of

data from Sudan on colorectal cancer

epidemiology. As a result, the purpose of this study

was to look at the MLH1 and MSH2 gene mutation

patterns in Lynch syndrome-associated colorectal

cancer in a series of Sudanese patients with CRC.

Medical Research Updates ⌠MRU⌡. ORIGINAL RESEARCH ARTICLE

MRU 2024; Vol.1(Issue2): 19-24 ISSN 200XX |

DOI: https://www.mruj.online/_downloads/c4d0aa9d3b9526980434be67dff8a1a8

MATERIALS AND METHODS

This study used 50 tissue blocks obtained from

histopathology facilities in El-Obeid, Norther

Kordofan State. Formalin-fixed wax-embedded

tissue blocks were acquired from CRC patients who

had surgical resections. All of the tissues were

formalin-fixed and wax-embedded. The sample

consisted of all CRC patients revealed to

histopathology laboratories in El-Obeid between

2017 and 2022. For the purpose of diagnosis,

conventional histology was used to make the main

diagnosis of CRC. In each case, a consultant

histopathologist confirmed the diagnosis of CRC.

Following that, immunohistochemistry methods

were used to detect two MMR proteins (MLH1 and

MSH2). Immunohistochemical testing was carried

out using two monoclonal antibodies (MLH1:

monoclonal mouse anti-human ki67, cloneMIB-1,

and MSH2: monoclonal mouse anti-human VEGF.)

as follows: Two formalin-fixed, paraffin-embedded

tumor slices (3m) were cut and mounted on

salinized slides (Dako). Following xylene

deparaffinization, slides were rehydrated in a

graded series of alcohol before being placed in

running water. Then, using a PT connection,

antigen retrieval for MLH1 and MSH2 was

performed. Endogenous peroxidase activity was

stopped for 10 minutes with 3% hydrogen

peroxidase and methanol, followed by 20 minutes

at room temperature in a moisture chamber with

100–200 l of primary antibodies, followed by a

washing in phosphate buffered saline. The MLH1

and MSH2 primary antibodies were ready for use

(Dako, Carpintera). After three minutes of washing

with PBS, the Dako-EnVision TM Flex kit incubated

dextran-labeled polymer for 20 minutes to detect

antibody binding. Finally, the sections were

washed three times in PBS before being stained

with 3 diaminobenzidine tetra hydrochloride

(DAB) (Dako) for up to 5 minutes to produce the

distinctive brown stain for the observation of the

antibody/enzyme combination. Hematoxylin was

then used to stain the slides. Positive and negative

control slides are also generated for each staining

session. The positive control slides included the

antigen under research, while the negative control

slides were made from the same tissue block but

treated with PBS rather than the primary antibody.

An investigator evaluated each slide, and a

consultant histopathologist validated and rated the

results.

Ethical Considerations

Beside obtaining ethical acceptance from relevant

authorized bodies, The Human Research Ethics

Committee (HREC) at the Prof. Medical Research

Consultancy Center has approved the research

protocol. HREC 0001/MRCC.12/23).

Statistical Analysis

The Statistical Package for the Social Sciences (SPSS)

version 23 was used for the statistical analyses.

Descriptive data reported as frequencies and

percentages were included in the statistical analysis.

RESULTS

Of the 50 patients, 23 (46%) were males and 27

(54%) were females, aged 20 to 75 years, with a

mean age of 48. Most patients were aged 41–60

years, followed by 61–75 and ≤ 40 years,

representing 23/50 (46%), 11 (22%), and 8 (16%),

respectively, as shown in Fig 1. The MLH1 mutation

was positive in 14 (28%) of the study population

and negative in 36 (72% of them). The MSH2

mutation was positive in 21 (42%), and negative in

29 (58%), of the study subjects. The MLH1 and

MSH2 mutations shared positivity in 9 (18%) and

negativity in 12 (24% of the cases), as seen in Fig 2.

Medical Research Updates ⌠MRU⌡. ORIGINAL RESEARCH ARTICLE

MRU 2024; Vol.1(Issue2): 19-24 ISSN 200XX |

DOI: https://www.mruj.online/_downloads/c4d0aa9d3b9526980434be67dff8a1a8

Figure 1. Description of the study subjects by age and mutation status.

Figure 2. Description of the study subjects by MLH1 and MSH2 mutation status.

DISCUSSION

LS is one of the most common hereditary cancer

syndromes in humans, affecting around 3% of

colorectal cancer patients who are not chosen, as

well as 10%–15% of persons who have mismatch

repair (MMR) genes, including MLH1, MSH2, MSH6,

and PMS2 [6, 7]. The reason we looked into MLH1

and MSH2 mutations in a group of Sudanese

patients with CRC was because pathogenic

germline variants in the MLH1, MSH2, and MSH6

Medical Research Updates ⌠MRU⌡. ORIGINAL RESEARCH ARTICLE

MRU 2024; Vol.1(Issue2): 19-24 ISSN 200XX |

DOI: https://www.mruj.online/_downloads/c4d0aa9d3b9526980434be67dff8a1a8

genes are thought to cause most LS cases [8]. MSH2

(42% of the mutations found in this study), MLH1

(28%), and shared mutations in 24% of the

subjects. Despite the fact that there is a scarcity of

data on the subject in Sudan, some studies have

revealed lower prevalence rates than ours. In a

Sudanese study, immunohistochemistry was

employed to determine the BRAF (V600E) mutant

status and mismatch repair (MMR) status. A

mismatch repair deficient (dMMR) subtype was

discovered in 16% of instances, and Lynch

syndrome (LS) was suspected in up to 14% of

patients [9]. The family traditions in North Sudan

are well-known for the practice of consanguineous

marriage. Certain Sudanese ethnic groups are

known to practice consanguinity and within-group

marriage, which are major contributors to the

community's elevated burden of genetic disease

[10]. In the current investigation, we noticed that

females were more frequently affected by CRC than

males. However, it has already been suggested that

patients with Lynch syndrome have a lifetime risk

of colorectal cancer (CRC) of 24-52%, which is

higher in males (28–75%) than in females (24–

72%). There is a clear genotype-phenotype

relationship, especially in the case of MSH6

mutations. With an MSH6 mutation, men have a

54% lifetime risk, while women have a 30% risk

[11]. There is a significant association (p < 0.001)

between the MSH6 mutation and female gender

and gynecological malignancies. Male MSH2 and

MLH1 carriers have higher rates of prostate, upper

GI tract, biliary, or pancreatic cancers compared to

the general population, whereas female carriers

have higher rates of endometrial and ovarian

malignancies [12]. In conclusion, according to the

present studies, CRC is more prevalent in women.

LS is more common in Sudanese patients with CRC

than in many other reports throughout the world.

MSH2 mutations are more prevalent in Sudanese

patients than MLH1 mutations.

Acknowledgment:

The authors would like to express their gratitude

to people at different histopathology laboratories

in El-Obeid city for their kind cooperation.

Authors contribution

BE: Conceptual, consultation, funding, and

approval of the final version.

SG: Conceptual, data analysis, funding, and

approval of the final version.

AAA: Conceptual, manuscript drafting, and

approval of the final version.

Funding: Self-funded.

Data availability: The data presented in this study

are available on request to the corresponding

author.

Disclosure of interest: No interest to declare.

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