LRP1B Polymorphisms Are Associated with Multiple Myeloma Risk in a Chinese Han Population.

Multiple myeloma (MM) is an extremely complex plasma cell malignancy that is genetically heterogeneous. A recent Genome-wide association study (GWAS) indicated that variation at 2q22 (rs61070260) influences MM risk. This association has not been validated to date in a Chinese Han population. In this study, we evaluated the association between rs61070260 in LRP1B and MM risk in a Chinese Han population involving 739 MM patients and 592 healthy controls. Our results indicated that rs61070260 in LRP1B was significantly associated with MM susceptibility (P=3.937×10-37). Furthermore, the linkage disequilibrium (LD) analysis of rs61070260 revealed an LD block encompassing exons 26, 27 and 28 of the LRP1B gene, and a subsequent sequencing analysis identified three SNPs (rs762074421, rs756168629, rs113600691) in exons 26 and 28 of LRP1B. For the SNP rs756168629 in exon 26, a missense mutation which results in a transition from arginine to histidine at position 1661 of the LRP1B protein, has not been found in Chinese populations according to the Chinese Millionome Database and Genome Aggregation Database (EAS), and this mutation was predicted to be deleterious or damaging by SIFT and PolyPhen. These findings firmly establish the role of LRP1B in contributing to MM susceptibility. In addition, the identification of a rare coding mutation (p.R1661H) in LRP1B detected in MM individuals was suggested to be harmful to the encoded protein, which was characterized as a candidate tumour suppressor; thus, LRP1B is likely to be a disease-associated gene that is implicated in the development and progression of MM.

Introduction

Multiple myeloma (MM) is a complex B-lineage neoplasm that accounts for 1% of all malignancies 1 and 1.9% of all cancer deaths 2. It is characterized by malignant plasma cell proliferation that is accompanied by supererogatory abnormal monoclonal proteins. MM exhibits considerable genetic heterogeneity, which can be divided into distinct groups such as hyperdiploid, hypodiploid, pseudodiploid and near-tetraploid MM, with a low or high prevalence of IgH translocations 3. Considerable progress has been made in the treatment of MM, but the aetiology underlying this malignant disease has not been clearly delineated to date 4, 5.

Genome-wide association studies (GWASs) have often focused on identifying significant genetic components for various complex human diseases, encompassing MM. A number of reported risk loci have the consistent effects of predisposing individuals to the development of MM 6. A recent GWAS confirmed that a polymorphism in 6q25.1 (rs12748648) was associated with MM and suggested an association at 2q22 (rs61070260) in LRP1B 7. LRP1B is composed of 91 exons, and the encoded protein is a multifunctional cell surface receptor that belongs to the low-density lipoprotein (LDL) receptor family 8-10. LRP1B was characterized as a candidate tumour suppressor, which significantly restrains cancer growth and invasion and, in particular, might modulate the amount of several cytokines in the tumour microenvironment through its endocytic activity 11. Deletions of LRP1B contributed to the low expression in several types of carcinomas, which was suggested to be associated with aggressive growth 9, 12-14.

The association between the identified risk loci of rs61070260 in LRP1B with MM has not been validated to date in a Chinese Han population. The aim of this study was to determine the allele and genotype frequencies of rs61070260 in the LRP1B gene in a Chinese Han population and to evaluate the significance of their impact on MM. Furthermore, most of the trait/disease-associated SNPs (TASs) typically identified by GWASs do not clearly identify causal variant that may be the TAS itself or a known SNP in strong linkage disequilibrium (LD) with the TAS 15, 16. It remains a major challenge to identify such causal variants. In this study, we simultaneously screened the exons in high LD with rs61070260 of the LRP1B gene to search for the potential causal variation for MM.

Materials and Methods

In this study, 739 MM patients and 592 healthy controls were used to replicate the association of rs61070260 in LRP1B with MM. Another group of 178 MM patients was used for identifying the potential causal variant(s) in LRP1B that in high LD with rs61070260. The ethics committees of the three hospitals approved the study, and informed consent was acquired from each participant.

Patient recruitment

The current study included 1331 participants (739 MM patients and 592 healthy controls) consecutively recruited from the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China), Beijing Union Medical College Hospital (Beijing, China) and Shengjing Hospital of China Medical University (Shenyang, China) between June 2015 and May 2017. The MM cases were diagnosed based on the bone marrow histopathological examination, clinical manifestations of serious end organ damage, including hypercalcemia, renal failure, anaemia and bone lesions (CRAB), and laboratory features 17. Basic demographic and clinical information, including age at diagnosis, sex, ISS stage, haemoglobin (Hb), serum albumin (Alb), serum calcium (Ca), serum creatinine (Crea), and β2-microglobulin (β2-MG), was collected. A total of 592 healthy controls that were free of monoclonal gammopathy of undetermined significance or MM were recruited from the groups of patients who had physical examinations in these hospitals. Blood samples (2 ml) were collected from each subject as a source of DNA.

Genotyping

The genomic DNA of 739 MM patients and 592 healthy controls was extracted by using a TIANamp Blood DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. Primer was designed using the Sequenom MassARRAY Assay Design 4.0 Software for the multiplex polymerase chain reaction (PCR) and for locus-specific single-base extension. PCR was conducted in a 384-well plate, and the products were subjected to locus-specific single-base extension reactions. After desalting, the products were dispensed onto a 384-format SpectroCHIP array (Sequenom), and genotyping was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The mass spectrograms and genotype data were analysed using MassArray Typer software version 4.0 (Sequenom) 18.

Statistical test for association

The statistical analyses were conducted using the PLINK tool set. Departure from HWE of rs61070260 frequency was assessed by the χ2 test in control subjects 19. Genotype and allele frequencies of the cases and controls were assessed using Fisher's exact test under the allele, dominant and recessive models and using logistic regression analysis under an additive model. The sex and age of the patients contributing the samples were used as covariates. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated, and P values less than 0.05 were considered to be statistically significant. Conditional logistic regression analyses were also performed for the associations of rs61070260 with the clinical characteristics of MM patients.

LD analysis

SNP genotype data of the HapMap CHB population were used to search for genomic regions in high LD (r2 > 0.8) with rs61070260 and the LD plot was constructed using the Haploview software.

Sequencing for causal variant(s)

Based on the results of the LD analysis, exons 26, 27, and 28 were sequenced in a new group of 178 patients with MM. Genomic DNA was extracted as previously mentioned and the primer sequences are listed in Table S1. Fragments were amplified and were subjected to Sanger sequencing. All sequencing chromatograms were compared with the reference sequence using Lasergene to detect the mutations.

The allele frequencies for the potential causal variants in current MM patients were compared to the Chinese Millionome Database (CMDB) and Genome Aggregation Database (gnomAD) (EAS) 20. The effect of the causal variant on the protein function was estimated by SIFT 21 and PolyPhen 22.

Results

Clinical Features of the Subjects

A total of 1331 participants, including 739 MM cases and 592 controls, were involved in this case-control study. Basic demographic and clinical information of the MM patients and controls are shown in Table 1. The average age was significantly lower for the 592 healthy controls than for the MM patients (P<0.001). This finding may be because MM is typical in elderly people, and over one-half of MM patients are older than 65 years at diagnosis 23, 24; in contrast, healthy controls were mainly recruited during their physical examinations, and these were mostly young men.

Table 1

Clinical features of subjects enrolled in this case-control study

Characteristics	Classification	MM Patients	Controls
Age, y (mean ± SD)		59.27 ± 10.11	47.83 ± 12.66
Sex, n (%)	Female	324 (43.84)	250 (42.23)
	Male	415 (56.16)	342 (57.77)
ISS stage, n (%)	I	45 (6.09)	-
	II	105 (14.21)	-
	III	184 (24.90)	-
Isotype (Immunoglobulin), n (%)	IgM	4 (0.54)	-
	IgD	21 (2.84)	-
	IgA	144 (19.49)	-
	IgG	312 (42.22)	-
	Light chain	176 (23.82)	-
	Nonsecretory	23 (3.11)	-
	NA	59 (7.98)	-
Isotype (Light chain), n (%)	κ	312 (42.22)	-
	λ	345 (46.68)	-
	Nonsecretory	23 (3.11)	-
	NA	59 (7.98)	-
Biochemical parameter (mean, range)	Ca (mmol/L)	2.17 (1.13-4.41)	-
	Crea (µmol/L)	115.29 (5.30-857)	-
	β2-MG (mg/L)	5.35 (0.73-38.5)	-
	Hb (g/L)	115.01 (29.8-169)	-
	Alb (g/L)	35.48 (2.70-51.00)	-

Abbreviations: Alb, albumin; β2-MG, β2-microglobulin; Ca, calcium; Crea, creatinine; Hb, haemoglobin; κ, Kappa; λ, Lambda; NA, not applicable.

Rs61070260 was strongly associated with MM susceptibility

No significant deviation from Hardy-Weinberg equilibrium (HWE) was observed for rs61070260 in the controls (P>0.05), and the case-control study showed a significant association between the SNP rs61070260 and MM risk, based on the result of the increased A allelic frequencies in MM patients (P=3.937×10-37) and clear differences in the genotype distribution in MM patients and healthy controls under the dominant model (AA+AG vs. GG: P=1.551×10-134), recessive model (AA vs. AG+GG: P=3.261×10-9), and additive model (AA vs. AG vs. GG: P=9.622×10-74) (Table 2). In the stratified analysis, no association was found between rs61070260 and any of the clinical features of MM patients (all P>0.05) (Table 3).

Table 2

Genetic association analysis of SNP rs16070260 in patients with MM and controls

SNP	Genetic model	Description	Cases/Controls	P value*
rs16070260(G>A)	Allele model	A	740/738	3.937×10-37
		G	308/876
	Dominant model	AA+AG	735/271	1.551×10-134
		GG	4/321
	Recessive model	AA	5/37	3.261×10-9
		AG+GG	734/555
	Additive model	AA	5/37	9.622×10-74
		AG	730/234
		GG	4/321

* The additive model was analysed using regression analysis, and the other model was analysed by Fisher's exact test.

Table 3

Association analysis of SNP rs61070260 with various clinical characteristics of MM patients

Characters of MM	Phenotypes	Number (P/N)	rs61070260 (G>A)
			F	χ2	P value	OR
Immunoglobulin Isotype	Heavy chain paraprotein	481/23	G (0.4979/0.500)	0.001188	0.9725	0.9917
	Light chain paraprotein	176/23	G (0.4915/0.500)	0.01777	0.894	0.9665
	Heavy vs. Light	481/176	G (0.4915/0.5021)	0.1159	0.7336	0.9585
Biochemical parameter	Crea (µmol/L)	148/554	G (0.4966/0.4991)	0.005728	0.9397	0.9901
	β2-MG (mg/L)	420/151	G (0.4975/0.4967)	0.0005958	0.9805	1.003
	Hb (g/L)	402/313	G (0.500/0.4984)	0.003593	0.9522	1.006
	Alb (g/L)	294/408	G (0.4983/0.4988)	0.0003087	0.986	0.9981

Abbreviations: N, patients negative for a certain phenotype; P, patients positive for a certain phenotype.

Candidate causal variation identified by Sanger sequencing

The LD analysis of rs61070260 revealed an LD block encompassing exons 26, 27 and 28 of the LRP1B gene (Figure 1). Therefore, Sanger sequencing of the three exons was conducted in another 178 MM patients to identify causal variations. We discovered three variant loci, rs113600691 (A>G) in exon 28 and rs762074421 (T>C) and rs756168629 (C>T) in exon 26 (Figure S1). Exon 27 was successfully sequenced, whereas no variant was found in this region. The SNPs rs113600691 and rs762074421 are located in the intronic region. The C allele frequency of rs762074421 and the G allele frequency of rs113600691 in MM patents were 0.0028 and 0.0112, respectively, which were lower than those in gnomAD (EAS) (Table 4). The SNP rs756168629 is a missense mutation located in the coding region (exon 26), which results in a substitution from arginine to histidine at position 1661 of the LRP1B protein. The frequency of the mutant T allele of this mutation in our MM patients was 0.0056. Particularly, according to the CMDB and gnomAD (EAS), this rare variant has not been found in Chinese populations to date. Furthermore, this variant is predicted to be deleterious or damaging by SIFT and PolyPhen. These findings provide original evidence of the role of the LRP1B gene in MM susceptibility and potentially its underlying pathogenesis.

Figure 1

Haplotype block of LRP1B containing rs61070260 based on 1000 genome CHB data. The standard Haploview LD colour scheme was based on D' and LOD (log of the likelihood odds ratio), and the number (divided by 100) in the small square represents the r2 value ranging from 0 (no linkage) to 1 (complete linkage). Haplotype blocks were defined using the confidence interval (CI) method. The red arrow indicates the position of the SNP rs61070260, which was strongly associated with MM risk.

Table 4

Mutant, SIFT and PolyPhen results for LRP1B

SNP ID	Localization	Exon	Number of patients (n/N)	Nucleotidechange	Amino acidchange	SIFT	PolyPhen	AF inMM patients	AF ingomAD (EAS)
rs762074421	chr2: 141598463	26	1/178	T>C	-	-	-	0.0028 (1/356)	0.0750 (3/40)
rs756168629	chr2: 141598619	26	1/178	C>T	R1661H	Deleterious	damaging	0.0028 (1/356)	0 (0/17220)
rs113600691	chr2: 141609507	28	4/178	A>G	-		-	0.0112 (4/356)	0.0220 (35/1622)

Abbreviations: AF, allele frequency; gnomAD (EAS), Genome Aggregation Database (East Asians); n, number of patients with certain gene mutation; N, number of patients involved in the sequencing.

Discussion

The results presented in the current study imply the strong association of the SNP rs61070260 in LRP1B and MM risk in Chinese Han population. The role of LRP1B polymorphisms in predisposition to pancreas cancer as well as non-malignant diseases, such as Alzheimer's disease, increased ventricular volumes in psychosis, preeclampsia and cardiovascular disease has been reported 25-28. A recent GWAS by Johnson et al. identified a suggestive association between rs61070260 and the risk of MM patients of European ancestry 7. The present study confirmed this association in a Chinese Han population, but further stratified analysis according to various characteristics of our MM patients, including immunoglobulin isotype and biochemical parameters (Hb, Crea, Alb, and β2-MG), showed no correlation with the SNP rs61070260.

Rs61070260 is located in the intron region of LRP1B, and it is likely to be functional or in linkage disequilibrium with another nearby functional variant. To discover the potential causal variants in LD with rs61070260 in MM, we performed an LD analysis of LRP1B. The results suggest that rs61070260 was located in an LD block encompassing exons 26, 27 and 28. Our sequencing analysis of 178 MM patients in these three exons revealed a rare missense variant (rs756168629, 4982 C>T), which causes a substitution of arginine to histidine in position 1661 and is predicted to be deleterious and damaging by SIFT and PolyPhen. It is highly possible that this rare variant may cause genetic defects in the LRP1B gene, leading to an increased susceptibility to MM.

LRP1B is characterized as a candidate tumour suppressor gene because the homozygous deletion of individual exons is observed in non-small cell lung cancer cell lines 29, 30. The protein encoded by the LRP1B gene is a multifunctional cell surface receptor that is a member of the LDL receptor family. The ability of the LRP1B protein to modulate the urokinase plasminogen system (uPA system) is thought to be a major underlying factor for the suppression of tumour invasion and metastasis. LRP1B displays a considerably slow rate of internalization of the uPA-PAI-1 complex, resulting in the subdued regeneration of unoccupied uPAR on the cell surface, which diminishes cell migration 31. The down regulation of LRP1B in renal cell cancer tissues and cell lines is frequently observed, and the depletion of LRP1B increased cell migration and invasion in vitro and in vivo 14, which strongly validated that LRP1B may function as a tumour suppressor. Hitherto, point mutations, homozygous deletions, and DNA methylation of LRP1B have been detected in multiple primary cancers 9. The variant that we detected in MM patients is damaging, indicating that it may contribute to the loss of LRP1B expression or be linked with specific clinical characteristics. Nevertheless, the prediction and deduction of the mutation remain to be verified.

The sample size of our study was limited, and large-scale population-based studies are warranted to validate our findings, especially the rare variant rs61070260 in MM patients. In addition, further functional studies that focus on delineating how variants of LRP1B identified in this study alter protein function and affect the development of MM are essential.

In summary, we confirmed the association between SNP rs61070260 in LRP1B and MM risk in a Chinese Han population and identified a rare mutation (p.R1661H) that may cause LRP1B protein defects. Further functional studies are required to fully understand the biological role of LRP1B in MM.

Supplementary figure and table.

Click here for additional data file.