Frequency of Variants in DNA-Repair Genes in a Southwest Colombian Population

Abstract

Objective To describe the frequency of mutations in DNA-repair genes in a southwestern Colombian population.

Methods We have designed an observational study, including 162 people from all ages from southwest Colombia. We have extracted and collected their DNA in filters. We have immersed the DNA in a phosphate buffer along with DNeasy package (Thermo Fisher Scientific, Waltham, MA, USA). The preparation process was with the TruSeq Exome Library Prep (Illumina, Inc. San Diego, CA, USA), then the obtained libraries were normalized with TruSeq Rapid Exome (Illumina, Inc. San Diego, CA, USA). We sequenced the full exome and identified the variants associated with 12 genes (ataxia telangiectasia mutated [ATM], BRCA1 DNA repair associated [BRCA1], BRCA2 DNA repair associated [BRCA2], checkpoint kinase 2 [CHEK2], epithelial cell adhesion molecule [EPCAM], homeobox protein Hox-B13 [HOXB13], mutS homolog 1, 2 and 6 [MLH1, MSH2, MSH6], nibrin [NBN], PMS1 homolog 2, mismatch repair system component [PMS2], and tumor protein p53 [TP53]). Descriptive statistics were performed with the R software (The R Foundation for Statistical Computing, Vienna, Austria).

Results A total of 7,315,466 pieces of data were sequenced in this population. The most frequently mutated genes were ATM (1,221 pieces of data; 13.2%), BRCA1 (1,178 pieces of data; 12.8%), BRCA2 (1,484 pieces of data; 16.12%), and NBN (965 pieces of data; 10.42%). The most common single nucleotide polymorphisms (SNPs) in these 12 genes were the following: BRCA2 (rs169547, rs206075, rs206076); ATM (rs659243, rs228589); TP53 (rs1625895, rs1042522, rs1642785); PMS2 (rs2228006, rs1805319); NBN (rs709816); and MSH6 (rs3136367)

Conclusion The BRCA2, ATM, BRCA1 and NBN DNA-repair genes were the most frequently mutated in this southwestern Colombian Population.

Resumen

Objetivo Describir la frecuencia de las mutaciones en los genes de reparación del ADN en una población del suroccidente de Colombia.

Métodos Diseñamos un estudio observacional que incluyó a 162 personas del suroccidente de Colombia de todas las edades. Hemos extraído y recogido el ADN en filtros. Los sumergimos en tampón fosfato junto con el paquete DNeasy (Thermo Fisher Scientific, Waltham, MA, EEUU). El proceso de preparación fue realizado con TruSeq Exome Library Prep (Illumina, Inc. San Diego, CA, EEUU); luego, las bibliotecas obtenidas se normalizaron con TruSeq Rapid Exome (Illumina, Inc. San Diego, CA, USA). Secuenciamos el exoma completo e identificamos las variantes asociadas a doce genes (ataxia telangiectasia mutated [ATM], BRCA1 DNA repair associated [BRCA1], BRCA2 DNA repair associated [BRCA2], checkpoint kinase 2 [CHEK2], epithelial cell adhesion molecule [EPCAM], homeobox protein Hox-B13 [HOXB13], mutS homolog 1, 2 and 6 [MLH1, MSH2, MSH6], nibrin [NBN], PMS1 homolog 2, mismatch repair system component [PMS2], y tumor protein p53 [TP53]). La estadística descriptiva se realizó en el programa R (The R Foundation for Statistical Computing, Viena, Austria).

Resultados Un total de 7.315.466 datos fueron secuenciados en esta población. Los genes más frecuentemente mutados fueron el ATM, con 1.221 datos (13,2%), el BRCA1, con 1.178 datos (12,8%), el BRCA2, con 1.484 datos (16,12%) y el NBN, con 965 datos (10,42%). Los polimorfismos de un solo nucleótido (PSN) más comunes en estos 12 genes fueron los siguientes: BRCA2 (rs169547, rs206075, rs206076); ATM (rs659243, rs228589); TP53 (rs1625895, rs1042522, rs1642785); PMS2 (rs2228006, rs1805319); NBN (rs709816) y MSH6 (rs3136367)

Conclusión Los genes de reparación de ADN BRCA2, ATM, BRCA1 NBN fueron los más frecuentemente mutados en esta población del suroccidente de Colombia.

Keywords

gene - prostate neoplasm - DNA - population - polymorphism

Palabras clave

gen - neoplasia de próstata - ADN - población - polimorfismo

Introduction

Prostate cancer (PCa) is the most frequent malignant neoplasia diagnosed in men, considering developing countries and the developed ones (excluding skin cancer); besides, it is one of the main causes of death.[1] [2] [3] This condition has a high incidence and prevalence; however, it is possible to find a substantial variation according to the geographic location and to some ethnic groups;[4] for example: in Asian countries like China, PCa has a lower incidence, while in North America, especially in Afro-American individuals, the incidence is higher.[5] [6] [7] Additionally, having a first-grade relative with PCa, the risk is increased by two, and with two relatives, the risk increases five times (OR 2 years 5 respectively).[8]

Another important factor is the general classification as sporadic versus familiar PCa,[6] being the latter the most frequently associated with the onset of the disease at early ages (∼45 years old) in members from the same family. Familiar Pca has been reported as frequent as 15%, while in sporadic PCa, the genetic material is damaged by environmental exposition to different factors during the lifetime (epigenetics), and its prevalence could be as high as between 80 and 90%.[6] [9] Prostate cancer is known as one of the most inherited cancers around the world (almost 50%); accordingly, it increases the autosomal dominant cancer predisposition.[10] [11]

Mutations in DNA-repair genes such as BRCA2 DNA repair associated (BRCA2), BRCA1 DNA repair associated (BRCA1), checkpoint kinase 2 [CHEK2], homeobox protein Hox-B13 (HOXB13), nibrin (NBN), and a series of single nucleotide polymorphisms (SNPs)[8] have been linked to PCa and have been used as biomarkers for PCa to predict clinical outcomes in a population.[8] [12] [13] [14] According to this intention, researchers need to continue studying genomic variants in different populations due to the pathological and genomic diversity.

Until now, there are no studies about genomic diversity regarding the risk for this inherited condition in a southwestern Colombian population (Nariño, Cauca, Putumayo, and Valle – which corresponds to 18% of the Colombian population). To consolidate the knowledge about this clinical condition, we need to translate information about mutations from similar predisposing populations to germline cancers to serve as sentinels to identify families with a high risk of developing this condition. The previous could serve to enable an early diagnosis in order to perform an intervention according to the individual genotype, which nowadays focuses on personalized medicine as a standard way to diagnose and to treat.

The objective of the present study was to describe the frequency of mutations in DNA-repair genes in a southwestern Colombian population.

Methods

We have designed an observational descriptive design that took place from 2014 to 2016. We have included people located in Southwest Colombia (Nariño, Cauca, Putumayo, and Valle) from all ages, multiracial, and coming from the public and private health systems. We have only excluded patients with cancer.

Sample Size

According to the expected frequency for hereditary PCa (∼15%), alfa 5% and an expected error of 5%, the calculated sample size was 162 people, and the sample selection was by convenience.

DNA Extraction

Each patient underwent blood extraction to obtain the DNA. All of the drops were collected in filter paper until they dried. These filters were immersed in phosphate buffer along with the DNeasy package (Thermo Fisher Scientific, Waltham, MA, USA). Each extraction was quantified and quality-verified to continue the sequencing processing.

Sequencing Protocol

The DNA aliquots from each sample underwent a preparation process with the TruSeq Exome Library Prep (Illumina, Inc. San Diego, CA, USA), then the obtained libraries were normalized to be sequenced using the TruSeq Rapid Exome (Illumina, Inc. San Diego, CA, USA). The normalized fragments with their corresponding adaptors for sequencing were charged in the HiSeq2500 system (Illumina, Inc. San Diego, CA, USA).

We sequenced the full exome and identified the associated variants, specifically the SNPs for 12 genes that have been associated with PCa (ataxia telangiectasia mutated [ATM], BRCA1, BRCA2, checkpoint kinase 2 [CHEK2], epithelial cell adhesion molecule [EPCAM], HOXB13, mutS homolog 1, 2 and 6 [MLH1, MSH2, MSH6], NBN, PMS1 homolog 2, mismatch repair system component [PMS2], and tumor protein p53 [TP53]).[15]

The present project accomplished all of the ethical international standards. Descriptive statistics were performed with the R software (The R Foundation for Statistical Computing, Vienna, Austria),[16] and the results are shown in frequency tables for each gene and its associated variants. We finally searched the variants in the following public databases: Exome Aggregation Consortium (ExAC),[17] PharmGKB,[18] Clinvar,[19] and Ensemble,[20] to look for some pattern to use the variants we found as markers.

Results

The Frequency of Mutated Genes

We obtained samples from 162 people from Southwest Colombia (aged between 18 and 65 years old). A total of 7,315,466 data were sequenced in this population. The most frequently mutated genes were ATM (1221 data; 13.2%), BRCA1 (1178 data; 12.8%), BRCA2 (1484 data; 16.12%), and NBN 965 data (10.42%) [[Fig. 1]].

Fig. 1 Frequency of genes associated with prostate cancer.

Associated Variants

The missense and stop variants associated with these 12 genes were present in 22% (2043 data) and in 0.13% (12 data), respectively [[Table 1]]. On the other hand, the ATM, BRCA1, BRCA2, and PSM2 genes were the most frequently associated with missense variants (247, 445, 490 and 289 data, respectively). Additionally, the BRCA2 and MLH1 genes had the highest frequency of stop variants (4 and 3 data, respectively) [[Table 2]].

Table 1 Frequency of variants associated with prostate cancer

VARIANT	Absolute frequency	Relative frequency (%)
Downstream	769	8.347
Frameshift	5	0.054
Intron	3906	42.397
Missense	2043	22.175
Nc_transcript	15	0.163
Splice	4	0.043
Stop	12	0.13
Synonymous	1727	18.745
Upstream	479	5.199
UTR	253	2.746

Abbreviations: Nc, Non-coding; UTR, Untranslated region.

Table 2 Frequency of variants by each gene associated with prostate cancer

Gene	Variant	Frequency	% by each gene	% by total variant
ATM	Missense	247	0.202	0.027
	Stop	1	0.001	0
BRCA1	Missense	445	0.378	0.048
	Stop	1	0.001	0
	Frameshift	4	0.003	0
BRCA2	Missense	490	0.33	0.053
	Stop	4	0.003	0
CHEK2	Missense	33	0.057	0.004
EPCAM	Missense	27	0.088	0.003
HOXB13	Missense	2	0.025	0
MLH1	Missense	67	0.16	0.007
	Stop	3	0.007	0.00
MSH2	Missense	13	0.025	0.001
	Stop	1	0.002	0
MSH6	Missense	70	0.077	0.008
	Stop	1	0.001	0
NBN	Missense	119	0.123	0.013
PMS2	Missense	289	0.323	0.031
	Stop	1	0.001	0
	Frameshift	1	0.001	0
TP53	Missense	240	0.365	0.026

Single Nucleotide Polymorphisms Associated with Prostate Cancer

The most common SNPs in these 12 genes were the following:

• BRCA2 (rs169547 [158 data; 97%], rs206075 [158 data; 97%], rs206076 [158 data; 97%]); ATM (rs659243 [158 data; 97%], rs228589 [134 data; 82%]); TP53 (rs1625895 [157 data; 96%], rs1042522 [141 data; 87%], rs1642785 [145 data; 89%]); PMS2 (rs2228006 [144 data; 88%], rs1805319 [142 data; 87%]); NBN (rs709816 [137 data; 84%]), and MSH6 (rs3136367 [136 data; 83%]) [[Table 3]].

• When comparing with bioinformatics databases, we found that rs169547 (BRCA2), rs 9534262 (BRCA2), rs659243 (ATM), rs228589 (ATM), rs1042522 (TP53), and rs2228006 (PMS2) had the higher Latino allele frequency found in ExAC. Additionally, rs1042522 (minor allele frequency [MAF] 0.46), rs228589 (MAF 0.46), and rs9534262 (MAF 0.47) coincided with the higher MAF found in Ensembl (https://www.ensembl.org/index.html). Additionally, rs799917 (MAF 0.46) and rs799905 (MAF 0.45) had a high MAF, although they were not found on ExAC [[Table 3]].

Table 3 Single nucleotide polymorphisms by gene (Only included single nucleotide polymorphisms with more than 100 data)

Gene	SNP	Frequency	% by population	% by gene	% by snp	Variant	Exome aggregation consortium			Clinvar	Ensembl
							Type	Total allele frequency	Latino allele frequency	Type	MAF	Highest MAF (1000 genomes)	Type
BRCA2	rs169547	158	97.53	0.118	0.02	T/C	Missense	0.9937	0.997	Benign	0.02	0.12	Missense
	rs206075	158	97.53	0.118	0.02	A/G	Synonymous	0.9931	NA	NA	0.03	0.13	Synonymous
	rs206076	158	97.53	0.118	0.02	G/C	Missense	0.993	NA	NA	0.03	0.13	Synonymous
	rs9534262	127	78.4	0.095	0.016	T/C	Benign	0.5208	0.51	Benign	0.47	0.5	Splice Region variant
ATM	rs659243	158	97.53	0.167	0.02	A/G	Missense	1	1	NA	0	0	Missense
	rs228589	134	82.72	0.141	0.017	A/T	Intron	0.572	0.6499	NA	0.46	0.49	Intron
	rs148973142, rs3092850, rs3218681, rs4987984	123	75.93	0.13	0.015	NA	NA	NA	NA	NA	NA	NA	NA
	rs2066734	121	74.69	0.128	0.015	TAA/T	Intron	0.4253	0.5695	NA	0.38	0.5	Intron
TP53	rs1625895	157	96.91	0.244	0.02	T/C	Non coding transcript exon	0.8613	NA	NA	0.17	0.35	Non coding transcript exon variant
	rs1642785	145	89.51	0.226	0.018	G/C	Non coding transcript exon	0.6689	NA	NA	0.42	0.5	5 prime UTR variant
	rs1042522	141	87.04	0.219	0.018	G/C	Missense	0.66	0.7115	NA	0.46	0.5	Missense
PMS2	rs2228006	144	88.89	0.172	0.018	T/C	Missense	0.8514	0.6563	Benign	0.12	0.33	Missense
	rs1805319	142	87.65	0.17	0.018	G/C	Synonymous	0.8109	NA	NA	0.17	0.35	Synonymous
	rs1805321	101	62.35	0.121	0.013	G/A	Missense	0.3854	0.3883	Benign	0.36	0.49	Missense
NBN	rs709816	137	84.57	0.147	0.017	A/G	Synonymous	0.4676	0.5891	Benign	0.39	0.49	Synonymous
	rs2308962	108	66.67	0.116	0.013	T/C	Splice region	0.3529	0.3481	Benign	0.38	0.5	Splice Region variant
	rs3736639	108	66.67	0.116	0.013	T/A	Intron	0.3535	0.348	Benign	0.38	0.5	Intron
	rs1805794	106	65.43	0.114	0.013	C/G	Missense	0.3453	0.3451	Benign	0.36	0.5	Missense
	rs1063045	105	64.81	0.113	0.013	C/T	Synonymous	0.3527	0.3477	Benign	0.38	0.5	Synonymous
	rs1061302	104	64.2	0.111	0.013	T/C	Synonymous	0.3451	0.3452	Benign	0.35	0.5	Synonymous
	rs2234744	103	63.58	0.11	0.013	G/A	Intron	0.3446	0.3447	Benign	0.35	0.5	Intron
MSH6	rs3136367	136	83.95	0.186	0.017	C/G	Intron	0.7426	0.6172	Benign	0.19	0.4	Intron
	rs2234731	122	75.31	0.166	0.015	NA	NA	NA	NA	NA	0.24	0.47	Intron
	rs2020911	108	66.67	0.147	0.013	A/T	Intron	0.4043	0.5252	Benign	0.4	0.5	Intron
EPCAM	rs1126497	123	75.93	0.469	0.015	T/C	Missense	0.5198	0.4629	Benign	0.33	0.5	Missense
CHEK2	rs5762757	121	74.69	0.303	0.015	A/C	Intron	0.5939	0.5648	NA	0.42	0.49	Intron
	rs5762756	113	69.75	0.283	0.014	NA	NA	NA	NA	NA	0.43	0.48	Intron
BRCA1	rs799917	113	69.75	0.105	0.014	G/A	Missense	0.41	0.3435	NA	0.46	0.5	Missense
	rs799905	112	69.14	0.104	0.014	G/C	Intron	0.4805	0.4468	Benign	0.45	0.5	5 prime UTR variant
MSH2	rs2303426	102	62.96	0.236	0.013	C/G	Intron	0.5047	NA	NA	0.37	0.5	Intron

Abbreviations: MAF, minor allele frequency; SNP: single nucleotide polymorphism.

Discussion

Race, diet, family history, environmental factors, and hereditary components have been reported as risk factors for PCa.[4] [5] [6] [7] Regarding the latter, genes such as BRCA1, BRCA2, MSH2, HOXB13, ATM, CHEK2, and NBN have been proposed as important candidates contributing to PCa.[8] [11] [12] [13] [14] The existence of SNPs for these genes further increases the risk, and even could be used as a prognostic biomarker for this condition.[21] Additionally, observing a germline mutation in some DNA-repair genes provides information to patients to look for counseling, to identify predisposition to cancer and, perhaps, to initiate interventions to reduce the risk.[11]

Regarding the frequency of these genes, Pritchard et al recently published a paper on patients with metastatic PCa from different series (the United Kingdom and the United States);[11] however, it is important to compare some of the results: the most frequently mutated gene was BRCA2 (5.35%) followed by CHEK2 (1.87%), and the 3^rd was ATM (1.59%). BRCA1 was present in 0.87% of the samples, while genes such as MSH2, NBN, MSH2, MSH6, and PMS2 had a lower frequency (0.14%, 0.29%, 0.14%, 0.14%, and 0.29%, respectively). The HOXB13 gene was not assessed in these series. These results are lower than ours, and we might expect to have a higher frequency, coming from data from metastatic patients. The incidence of PCa, at least in Cali, Colombia, has been stable during the last few years (2002 to 2007; Annual Percent Change [APC]: 0.5%), the mortality diminished,[22] and the population of afrodescendants increased from 15% in 1964 to 26% in 2005.[23] However, these data do not explain these results, since there are no previous studies that described the frequency of these genes and their variants.

Pilié et al[24] published another important study to be discussed. This study was not about the frequency of genes, but it identified genes associated with PCa and other concomitant tumors (at least one additional tumor). They also found similar germline genes: BRCA2, ATM, MLH1, BRCA1, CHEK2, and HOXB13, although they found 10% of pathogenic or likely pathogenic mutations in cancer-predisposing genes. They found 525 missense, 7 frameshit, 5 in-frame coding insertion or deletions, and 2 nonsense variants (rs80359440, rs80359515, rs61757642, rs587781658, rs373226793, rs180177143, rs759113408, rs17879961, and rs138213197). In contrast, in the present study, we found a higher frequency of this type of variants and none of the SNPs they reported.

In our study, which is identified as the first study of this type in southwestern Colombia, the BRCA2 gene was the most frequently mutated, and it has been considered one of the few genes that confer a high risk of suffering from PCa and other conditions.[3] [6] [25] [26]

The BRCA2 gene has an important role in the repair of double-strand DNA breaks, functioning by regulating the intracellular transport and the activity of RAD51, a critical protein in homologous recombination.[27] [28] [29] The most frequent variants found for this gene, such as rs169547, rs206075, rs206076, and rs9534262, are similar to those found in other studies.[30] [31] These have been considered benign mutations[32] [33] [34]–the first three were reported in Colombia by Arias-blanco et al[35]–and rs9534262 was considered a deletion-type mutation with an unknown clinical relevance according to D'Argenio et al,[33] although it is registered as benign according to ClinVar. Rs9534262 seems to be a quantitative trait locus (QTL) of significant importance, since it regulates the levels of the alternative transcripts of the BRCA2 gene.[36]

The 2^nd most commonly mutated gene was ATM, which exerts control in cell division. When detecting damage in the genetic material, it leads to either cell cycle arrest, to DNA repair, or to apoptosis.[37] [38] [39] In addition, it can be considered as the main transducer in the repair process of the rupture of the double-strand, in which it recruits and cooperates with other sensor proteins, such as 53BP1 (p53 binding protein) and BRCA1.

The BRCA1 gene is the third mutated gene found and associated with an increased risk of sporadic PCa (3.5 times), while for germline mutations in this gene, while only 0.44% of PCa for germline mutations.[5] [40] [41] The Breast Cancer Linkage Consortium (BCLC) described that men aged 65 with mutations in the BRCA1 gene report an increase in the risk of PCa with a relative risk (RR) of 1.82, being linked to a series of cellular processes such as response and repair of DNA damage,[5] [39] [41] [42] transcriptional regulation, and chromatin modeling. This is the key in cellular control systems to be involved in all of the phases of the cycle, in such a way that it can block cell proliferation and promote the apoptosis of cells with a high risk of malignant transformation.[5] [41] For this gene, the most frequent variant was rs799917, a nonsense mutation-type polymorphism,[33] but benign according to Arias-blanco et al.[35] This one is located in the coding sequence of the gene and affects the interaction of miR-638 with the mRNA of the BRCA1 gene.[43] It is also associated with the risk of suffering from breast, stomach, and esophagus cancer, additionally.[43] [44] [45] These two genes have not been reported in Colombia, according to the literature review.

Another important gene found in the present study was NBN, which is a component of the protein complex hMRE11/hRad50/NBN that participates in the initiation of a response to DNA damage and is linked to the repair of double-strand rupture.[46] [47] [48] This acts in the nonhomologous junction pathway as a DNA damage sensor and in the homologous recombination pathway, participating in DNA repair and in the cell cycle checkpoint in the S phase.[48] The rs709816 polymorphism is one of its most frequent variant, which has been associated with bladder cancer[48]; another one was rs3736639, which has been found at a slightly higher frequency in patients with leukemia.[46]

On the other hand, the rs1805794 polymorphism presents positive association with different types of cancer, such as bladder,[49] [50] lung,[51] breast, nasopharyngeal,[52] [53] [54] osteosarcoma[55] and PCa. In this last one, GG carriers have an almost twice increased risk of developing PCa, suggesting a role of NBN in the diagnosis and in the progression of PCa.[46] The rs1061302 polymorphism has been associated with an increased risk for lung, larynx and liver cancer. Single nucleotide polymorphisms might affect the proper binding of the complex and thus alter its ability to repair or detect DNA breaks.[48] [51] Although this variant is considered a synonym polymorphism, the altered nucleotide may affect the stability of the mRNA, the splicing, or the transduction rate.[48]

Additionally, rs169547 (BRCA2), rs9534262 (BRCA2), rs659243 (ATM), rs228589 (ATM), rs1042522 (TP53), and rs2228006 (PMS2) had the higher Latino allele frequency, and some of them showed coincidence with a higher MAF. This would be the first description of these kinds of SNPs for the Colombian population that leads to studying the way to work with them as a network as biomarkers for PCa.

Strengths and Limitations

This is the first study performed in the southwestern Colombian population, which is extremely important to characterize our population and to begin to recognize the risk for developing PCa. The quality of the samples, the methods we followed, and the quality and the analysis performed with the data are other important issues to highlight from our study.

Regarding the limitations, we could declare that most of the variants found in the present study are considered as benign. Nonetheless, it is important to go deeper to analyze longitudinally these data and to obtain the real risk in this population. Perhaps another important limitation could be that the extrapolation of these data is limited to people with similar characteristics in southwest Colombia.

Conclusions

To conclude, the BRCA2, ATM, BRCA1, and NBN DNA-repair genes were the most frequently mutated in this southwestern Colombian population. Additionally, there were nonpathological variants, such as rs169547, rs206075, rs206076, and rs9534262, and some other variants, such as rs799917, rs3736639, rs1061302, and rs1805794, which were associated with the NBN gene, which is highly linked to PCa.

Conflicts of Interests

The authors have no conflicts of interests to declare.

Declaration Section

The authors declare that the present study did not receive any funding.

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