慢性淋巴细胞白血病免疫球蛋白重链可变区突变特点分析

目的探讨慢性淋巴细胞白血病(CLL)患者的免疫球蛋白重链可变区(IgHV)突变和未突变患者的实验室检查特点。方法对2020年2月至2021年2月天津见康华美医学诊断中心266例行IgHV基因检测的CLL初诊患者的实验室检查结果进行总结, 分析IgHV基因突变状态、基因片段的使用特征;对突变与未突变患者的免疫表型、基因突变筛查、染色体核型及荧光原位杂交(FISH)结果进行差异性比较。结果患者男性172例, 女性94例, 中位年龄67岁(20～82岁)。(1)IgHV突变患者比例高于IgHV未突变患者(69.2%∶30.8%)。(2)VH家族以及基因片段的使用均存在明显的偏颇性:VH3家族(142/266, 53.4%)、VH4家族(75/266, 28.2%)和VH1家族(34/266, 12.8%)的发生率总和约为95.0%;具体到基因片段, 使用VH4-34(26/266, 9.8%)、VH3-23(25/266, 9.4%)、VH3-7(24/266, 9.0%)、VH4-39(16/266, 6.0%)的比例总和约为35.0%, 其中VH3-20片段和VH3-49片段仅发生于Ig...     

BIOMED-2引物系统检测急性淋巴细胞白血病患者免疫球蛋白基因重排

目的 探讨BIOMED-2引物系统检测成人急性淋巴细胞白血病(ALL)患者Ig基因重排的敏感性,分析Ig基因重排方式、各胚系基因的利用频率等.方法 采用BIOMED-2引物系统扩增29例成人ALL患者Ig重链(IgH)和Ig轻链(IgL)重排基因.将PCR产物直接测序,使用IMGT/V-QUEST等生物信息资源分析B细胞-ALL(B-ALL)患者IgH和IgL基因重排类型、胚系基因片段利用及体细胞突变情况.结果 24例B-ALL患者中IgH完全重排阳性率为70.8%,不完全重排为12.5%,IgK VH-JH重排为29.2%,IgK删失元件(IgK-Kde)重排为25.0%,未检测出IgL阳性重排.所有B-ALL患者Ig基因重排检出率可达100%.5例T细胞-ALL(T-ALL)患者1例检测到IgH不完全重排阳性,2例IgK VH-JH重排阳性,2例未检测出Ig基因重排.B-ALL中IgH重排优先利用的V、D、J家族分别为VH3和VH4、DH3和JH6.5例IgK重排中4例利用了 Vκ1家族.重排基因中发生替代突变的基因占23.5%.替代突变零星散布于Ig基因全长,互补决定区中替代突变与静寂突变的比例<1.结论 BIOMED-2引物系统可以检测出绝大多数B-ALL患者的Ig基因重排,国内的临床检测资料亦然.这是一种有效的检测工具,为定量分析微小残留病(MRD)水平提供了基础资料.     

骨髓瘤细胞免疫球蛋白重链基因可变区的研究

为分析多发性骨髓瘤 (multiplemyeloma,MM )细胞对免疫球蛋白重链基因可变区 (VH)基因家族的取用 ;根据VH 基因突变特点 ,揭示MM细胞的起源。以重链基因可变区 (VH1 VH6 )基因家族特异性引物 ,用PCR法扩增骨髓瘤细胞系CZ 1细胞和 98例MM患者外周血单个核细胞VH 基因片段 ,纯化后的PCR产物和pMD18 T载体连接并转化JM10 9细菌 ,经克隆鉴定后 ,目的DNA片段用末端双脱氧法测定DNA序列 ,和其对应的胚系基因序列比较。结果表明MM细胞对各VH 基因家族的取用顺序为VH3>VH1>VH4 >VH2 >VH5 >VH6 ;MM细胞VH 基因互补决定区 (CDR )氨基酸替换性突变 (R突变 ) /氨基酸静寂性突变 (S突变 )等于 9 6 7,而骨架区 (FR )R/S等于 0 87,而且随着疾病的进展 ,IgVH 基因并不发生进一步的突变。结论是MM前体细胞在进行VDJ基因重排时 ,对VH 基因家族的取用和基因家族相对大小有关 ;MM细胞可能起源于已经发生抗原选择和体细胞突变的B记忆细胞或前浆细胞。     

山东半岛地区家族性和早发性乳腺癌BRCA2基因突变研究

目的 研究山东半岛地区家族性和早发性乳腺癌中乳腺癌易感基因BRCA2的突变位点及携带情况.方法 应用PureGene DNA纯化系统提取52例家族性和早发性乳腺癌患者的外周血单核细胞DNA,对BRCA2基因的全部编码序列及内含子和外显子拼接区进行扩增,扩增产物用变性高效液相色谱进行初筛,对发现异常片段经重新扩增后进行DNA测序证实.结果 在52例乳腺癌患者中发现3个(5.8%)BRCA2的致病性突变(2001delTTAT,4099＞T,5873C＞A).其中,家族性乳腺癌突变率达到12%(3/25),在单纯早发性乳腺癌病例中未发现致病性突变.结论 在家族性乳腺癌患者中,BRCA2基因突变可能具有重要作用,在此人群中有必要进行相关的基因检测.     

性反转综合征患者SRY、WT1及SOX9基因检测

目的探讨性反转综合征的发生与性别分化相关基因SRY、WT1及SOX9之间的关系。方法收集4例46,XY女性患者,1例46,XX男性患者及18例核型性别一致的正常人,进行染色体核型分析、Y染色体微缺失、PCR及Sanger测序检测。结果 4例46,XY女性患者Y染色体无片段缺失,SRY基因阳性且无突变,3例患者的WT1基因8号外显子(CHM11:32396408)发现纯合同义突变(AG),1例患者的SOX9基因3号外显子检测出SNP(CHM17:72124410,AC)。46,XX男性患者Y染色体微缺失结果:SRY、ZFY、s Y123和s Y127基因出现目的条带;且SRY基因无突变。结论本研究中,4例46,XY女性患者无SRY基因突变;WT1基因的突变和SOX9的SNP不是造成性反转综合征的常见原因。除了SRY、WT1和SOX9基因之外,还有其它基因的突变可以造成XY患者性反转。SRY基因阳性是XX患者发育成男性的原因。     

应用聚合酶链反应-限制性片段长度多态技术快速检测中国耳聋人群基因突变热点

目的 通过筛查耳聋基因热点突变:GJB2基因的235delC、SLC26A4基因的IVS7-2A>G和线粒体12S rRNA(12S)基因1555 A>G达到快速诊断耳聋患者.方法 多重PCR扩增包括GJB2、SLC26A4及12S基因的3个片段,限制性片段长度多态分析是否存在相应位点的突变.结果 200例耳聋患者中,共检测出235delC纯合突变18例,杂合突变18例;IVS7-2A>G纯合突变2例,杂合突变13例;1555 A>G突变8例.检测结果均与测序结果相符合.3个热点突变基因的致病单体的检出率为21.7%,基因诊断率为14%.结论 应用聚合酶链反应-限制性片段长度多态技术检测耳聋患者的热点突变是一种快速、简便、高效、经济的耳聋致病基因筛查的方法.     

变性高效液相色谱法分析非综合征型耳聋人群SLC26A4基因突变

目的 研究非综合征型耳聋(nonsyndromic hearing loss,NSHL)患者SLC26A4基因的突变情况,为临床上NSHL患者基因诊断提供指导.方法 PCR分别扩增SLC26A4基因的21个外显子及其侧翼序列,所得目的 片段用变性高效液相色谱(denaturing high-performance liquid chromatorgraphy,DHPLC)进行突变筛查,有异常峰形的样本进行DNA测序.结果 在所选30例无血缘关系且GJB2基因检测未发现突变的NSHL患者中,共检测出10种SLC26A4基因变异,其中包括7种已知突变,2种未见报道的新突变(F572L和D87Y),及一种已知多态(Ivs11+47T＞C),其中Ivs7-2A＞G是最常见的突变,约占总突变的40%.结论 SLC26A4基因为仅次于GJB2的导致NSHL的相关基因,在(GJB2基因检测未发现突变的NSHL人群中SLC26A4基因的检出率达到23.3%,其中Ivs7-2A＞G是其最常见的突变.     

家族性先天性珊瑚状白内障患者基因突变筛查

目的 检测一家系3例先天性珊瑚状白内障患者相关基因的致病性突变位点.方法 对一家系3例患者及4名正常家族成员进行了详细的病史采集和常规眼科检查,采血从白细胞中提取基因组DNA,PCR扩增功能候选基因或基因片段,直接双向测序,Blast比对筛查突变.结果 CRYAA、CRYAB、CRYBB2的第6外显子、CRYGD、CRYGC、GJA8、GJA,和MIP基因测序结果除发现了已经报道的单核苷酸多态性(single nucleotide polymorphism,SNP)外未发现致病性基因突变.结论 该家族患者并非由CRYGD以及其他的常见先天性白内障功能候选基因的已知突变引起,证实珊瑚状白内障存在遗传异质性.     

甲状腺乳头状癌中BRAFV599E点突变与RET/PTC融合基因的检测

目的检测甲状腺乳头状癌(PTC)及其他类型甲状腺良恶性肿瘤中BRAFV599E的点突变及 RET/PTC1、3融合基因的表达状况,探讨二者与PTC临床病理学特征的关系.方法用聚合酶链反应(PCR)及逆转录(RT)-PCR分别检测95例石蜡与新鲜甲状腺病变组织中BRAFV599E点突变和RET/PTC1、3融合基因.结果 (1)仅在PTC中检测到BRAFV599E的突变,突变率56%(37/66),在经典型PTC和高细胞型PTC中突变率分别为70%(29/41)和2/3,在滤泡型PTC及其他类型甲状腺病变中未检测到BRAFV599E的突变.统计学分析BRAF突变与性别、年龄、伴慢性淋巴细胞浸润及淋巴结转移无明显关系(P>0.05).(2)PTC中RET/PTC检出率21.2%(14/66),其中5例RET/PTC1阳性(7.6%),9例RET/PTC3阳性(13.6%).RET/PTC融合基因阳性的14例PTC中未检测到BRAFV599E突变.其余29例良恶性病例中未检测到RET/PTC融合基因.RET/PTC融合基因的表达与PTC的临床病理学特征无明显关系(P>0.05).结论 (1)BRAFV599E突变和RET/PTC融合基因是PTC较特征性的遗传学改变,可作为PTC诊断和鉴别诊断提供分子学的依据,BRAFV599E突变可能是甲状腺乳头状癌表型的重要决定因素之一;(2)BRAFV599E突变与PTC的经典型和高细胞型两种主要亚型密切相关;(3)BRAFV599E突变与RET/PTC融合基因可能在PTC中是独立事件.     

中国人家族性腺瘤性患肉病患者结肠腺瘤性患肉病基因的胚系突变

目的 探讨中国人家族性腺瘤性息肉病(familial adenomatous polyposis,FAr)患者的结肠腺瘤性息肉病(adenomatous polyposis coli,APC)基因的胚系突变类型.方法 对9个FAP家系18名成员进行多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MLPA)检测APC基因有无大片段缺失.再应用PCR扩增APC基因的15个外显子区域,经变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)对每个扩增片段进行筛查,流出峰异常的片段,经DNA测序验证小片段的改变.结果 9个家系中有3个家系发现有APC基因的胚系突变:家系2为c.3184-3187 del CAhA,家系4为c.5432C＞T,家系9为c.3925-3929 del AAAAG.3种突变中c.5432C＞T在数据库中未见报道.结论 中国人不同的APC基因的胚系突变可引起FAP;无APC胚系突变的FAP患者的发病可能存在其他的机制.     

Pattern and distribution of immunoglobulin VH gene usage in a cohort of B-CLL patients from a Northeastern region of Italy.

We analyzed individual VH gene rearrangements in 55 consecutive B-chronic lymphocytic leukemia (B-CLL) patients collected from a northeastern region of Italy, stressing the possible differences related to geographic characteristics of the cohorts studied. Considering the percentage of somatic mutations present in the VH gene sequences and using the 98% cut-off value, 38 of the 55 B-CLL (69%) patients displayed somatic hypermutations and 17 (31%) had a germline configuration. Our results confirm and extend the observations of a bias in the use of certain VH, DH, and JH genes among B-CLL cells. The most frequently used VH genes were VH1-69 (12.7%) with VH3-23 (12.7%) and VH4-34 (10.9%). Collectively these genes accounted for 36.3% of the cases. In the mutated cases, the range of mutations varied from 2% to 15%, with a median of 6.5%. VH1-69 (7 cases, all unmutated) carried few mutations as opposed to VH3-23 (7 cases, 5 of which mutated), VH4-34 (6 cases, all mutated), and VH3-30 (5 cases, all mutated), which show a high load of mutations. D3 family genes were found frequently (38.1%) followed by D2 (27.2%) and D6 (18.1%). The individual D segment most frequently used was D3-3, which was present in 16.3% of cases. There was predominance of the JH4 gene (49%) followed by JH6 (40%). Analysis of the distribution of replacement and silent mutations in the mutated sequences using the method of Lossos showed in 39.4% of cases evidence of antigen selection in the framework region and/or complementary determining regions. In comparison with a recent study on B-CLL patients from the Mediterranean area, the VH4-34 gene was significantly overused in the mutated group at a percentage double that of the Italian cohort reported in this study (10.9% vs. 5%), but at a frequency similar to the entire Mediterranean region (10.7%). We also found an over-representation of VH1-69 usage in the germline group, at a frequency (12.7%) higher than previously described by the same authors (Italian 8%, Mediterranean 10.7%). On the contrary, VH3-07 and VH3-49 were not much used in our study (5.4% and 1.8%, respectively) compared with the Italian group (8% and 5.1%). In our study, VH3-23 gene segment was frequently expressed, at frequency as high as that of VH1-69, a finding in keeping with reported B-CLL Italian data, but higher than the entire series of the Mediterranean area (12.7% vs. 9.2%); VH3-21 gene, frequently expressed in northern European CLL but rarely in the Mediterranean area, was completely absent. This biased usage of VH family genes may reflect a geographic leukemic repertoire, perhaps owing to a peculiar genetic background, depending on variations in germline composition of the IgVH locus or to the effect of a potential environmental element less frequently encountered in different regions.     

Extensive allelic sequence variation in the J region of the human immunoglobulin heavy chain gene locus

During the initial stages of B lymphocyte differentiation heavy chain variable (VH), diversity (DH) and joining (JH) gene segments recombine to form a functional heavy chain variable region (VDJ) gene. Evidence for genetic polymorphism of the human JH gene segments has been obtained from mature rearranged VDJ sequences. We conducted an analysis of the published rearranged JH gene sequences and found that the JH alleles present in the two published germ-line JH region sequences were rare (approx. 2%) in the rearranged sequences. As an attempt to explain this discrepancy a 2.5-kb strech of DNA containing all the six heavy chain JH region genes and the most 3' DH gene segment, DHQ52, was amplified by the polymerase chain reaction from 39 individuals and analyzed for restriction fragment length polymorphism. Five new JH region haplotypes were found and sequenced. These new haplotypes contained the coding segment alleles that were frequent in antibody genes. Surprisingly, a high number of interallelic differencies in the non-coding sequence was found between the new and the two previously published haplotypes implying that the haplotypes had been separated early in evolution. In this respect the JH locus resembles HLA loci.     

Characterization of a human monoclonal autoantibody directed to cardiolipin/beta 2 glycoprotein I produced by chronic lymphocytic leukaemia B cells.

We determined the specificity and sequence of immunoglobulin molecules synthesized by monoclonal B cells from a patient with chronic lymphocytic leukaemia (CLL) who presented with a number of clinical and biological autoimmune symptoms. Heterohybrids obtained by fusion of CLL cells with the mouse X63-Ag 8.653 myeloma produced IgM lambda MoAbs directed to the cardiolipin/beta 2 glycoprotein I (beta 2GPI) complex and ssDNA. They were devoid of polyreactivity. Nucleotide sequence analysis of the variable domain of the mu chain indicated the utilization of the VH4 71.2 gene or one allotypic variant, DXP4 and JH3 segments. The lambda light chain used the single gene from the V lambda 8 subfamily, J lambda 3 and C lambda 3 genes. The VH gene displayed 11 nucleotide changes in comparison with its putative germline counterpart. However, these nucleotide changes correspond to variations observed in other published VH4 sequences, suggesting gene polymorphism rather than somatic mutation. DXP4 and JH3 were also in germline configuration. The VL gene exhibited a single replacement mutation in CDR1. These data suggest that the monoclonal CLL B cells in this patient retained VH and VL genes in germline configuration although they secreted a pathogenic anti-cardiolipin antibody associated with clinical symptoms, vasculitis and thrombosis, which may be provoked by antibodies to the phospholipid/beta 2GPI complex.     

Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire.

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.     

Immunoglobulin gene sequence analysis to further assess B-cell origin of multiple myeloma.

To further characterize the B-cell origin of multiple myeloma, our laboratory performed immunoglobulin gene sequence analyses of four cases of myeloma (three immunoglobulin A and one immunoglobulin G). Three tumors expressed VH3 genes and one expressed a VH1 gene, while the light chains included two V lambda and one V kappa III; one light chain was not isolated. The closest homology to published germ line genes ranged from 91 to 97%. In two cases, the expressed VH genes were compared with the putative germ line precursor VH genes isolated from autologous granulocyte DNA and appeared to have mutated randomly from the germ line gene. By sequencing multiple clonal isolates from each tumor sample, we found no evidence for ongoing mutation in three cases; in one case, however, clonotypic heterogeneity was evident. The analysis of DH- and JH-region genes revealed (i) limited or absent N nucleotide insertions (two of four cases), (ii) the presence of a DH-JH junction resulting from sequence overlap between the DH and JH genes (one of four cases), (iii) the absence of somatic mutations (two of four cases), and (iv) restricted JH gene usage of a JH6 polymorphism (three of four cases). These analyses of DH and JH genes suggest that multiple myeloma, similar to what has been proposed for chronic lymphocytic leukemia, may derive from B cells which have rearranged during fetal development rather than during adult life.     

Consensus primers for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell lymphomas

AIMS: To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS: Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS: Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION: The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.     

Diversity of immunoglobulin heavy chain gene segment rearrangement in B lymphoblastoid cell lines from X-linked agammaglobulinemia patients

X-linked agammaglobulinemia (XLA) is characterized by an arrest in early B lymphocyte differentiation. Precursor B cells are present in the bone marrow (BM), whereas peripheral blood B cell numbers are severely decreased. A series of Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) was established from peripheral blood of three XLA patients belonging to one pedigree. These BLCL manifested productive VHDJH rearrangements and a random utilization of the VH families. The CDR3 regions of the rearrangements varied in length from 12 to 47 nucleotides and included N regions in all cases. The results supported the conclusion that the few B lymphocytes in peripheral blood of XLA patients exhibit all mechanisms that generate immunoglobulin (Ig) heavy (H) chain diversity. However, no evidence for somatic mutation was found. Within the VH3 family 50% of the expressed VH gene segments belonged to a single subgroup and within the VH4 family a preferential utilization of one VH4 gene element was observed. The utilization of H chain joining (HH) elements was biased to JH4 and JH6 and a high percentage of the CDR3 regions was found to be generated by unconventional mechanisms, such as multiple D usage and the fusion of D elements to D segments with irregular recombination recognition signals. These unique features of the recombined and expressed VHDJH regions in XLA may explain the inability of XLA patients to respond to a variety of antigens. Alternatively, they could be secondary to a B lymphocyte maturation defect in XLA.