1、一11β-羟化酶缺陷症家系分子致病机制研究 2、促甲状腺激素对冠心病患者血脂水平的影响研究
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摘要
研究背景：先天性肾上腺皮质增生症(congenital adrenal hyperplasia, CAH)是内分泌系统最常见的遗传性疾病之一,属于常染色体隐性遗传性疾病,由皮质激素合成酶基因突变致肾上腺皮质类固醇激素合成障碍所引起。
CAH是由于皮质类固醇激素合成过程中任何一种酶先天性缺陷,引起肾上腺皮质束状带合成的皮质醇完全或部分缺乏,经下丘脑-垂体-肾上腺轴反馈调节,引起促肾上腺皮质激素释放激素(CRH)和促肾上腺皮质激素(ACTH)分泌增加,导致了肾上腺皮质增生,另一方面,某一酶的缺陷也使得皮质醇生物合成链中缺陷酶近端的前体产物因不能继续向下游合成而堆积,和雄激素的过度生成。CAH临床主要特点为肾上腺皮质功能不全,性腺发育异常,伴或不伴水盐代谢紊乱,高血压。本症表现出女性男性化或性幼稚,男性假性性早熟或女性化,失盐或非失盐,高血压等一系列症状。
典型CAH的发病率约为1／10000,而非典型的发病率约为典型的10倍,因其临床表现错综复杂,且对疾病认识及实验室检查技术的不足,诊断方面尚存在许多困难,一般需要从临床表现,实验室检查和基因分析三方面进行综合诊断。目前CAH基因缺陷的相关检测技术没有推广,在我国临床工作中尚未常规开展。
11β-羟化酶缺陷症(steroid11β-hydroxylase deficiency,11-OHD, OMIM:+2020101是CAH中第二大类型,约占5-8%,发病率约为1／100,000至1／200,000。11β-羟化酶缺乏引起皮质醇合成减少,对ACTH的反馈抑制减弱,使下丘脑和垂体分泌的CRH及ACTH增加,导致束状带产生更多的皮质醇前体。这些前体物质通过旁路代谢,在网状带产生大量雄激素,导致高雄激素血症。此外,11-去氧皮质酮和11-去氧皮质醇水平升高,由于它们具有类盐皮质激素的作用,从而引起水钠潴留、高血压及低钾血症。11β-羟化酶缺陷症的临床表现包括经典型和非经典型。经典型11β-羟化酶缺陷症患者可表现为女性外生殖器男性化,男、女患者都可出现的假性性早熟,由于生长过快骨骺愈合提前引起身材矮小,以及血压升高、血钾降低。非经典型在女性患者多表现为月经紊乱,多毛和痤疮,类似多囊卵巢综合征的表现；在男性患者可无症状。
由于11β-羟化酶缺陷症的发病人数较少,其分子遗传学研究相对较少。现已证实：11β-羟化酶缺陷症的分子遗传学缺陷是由于CYP11B1基因突变所致。CYP11B1基因(GeneID:1584; MIM:610613; GenBank:NC_000008.10)位于第8号染色体长臂上的21区(8q21),由9个外显子组成(见图1-6),长度为6.03Kbp,编码503个氨基酸残基。
CYP11B1基因编码11β-羟化酶,该酶属于细胞色素P450蛋白超家族中的一员,在肾上腺束状带表达,位于线粒体内膜,催化11-去氧皮质醇和11-去氧皮质酮分别转变为皮质醇和皮质酮。11β-羟化酶分子量约47kDa,是一种含有血红素的蛋白,具有依赖还原型烟酰胺腺嘌呤二核苷酸磷酸(NAPDH)的氧化还原体系,该体系由黄素蛋白——皮质铁氧还蛋白还原酶及铁硫蛋白——皮质铁氧还蛋白构成。细胞色素P450蛋白家族的成员结构高度保守,其核心结构由一个被称作“曲”的螺旋,一个由J螺旋和K螺旋组成的四螺旋束以及两套β片层构成。该结构构成了血红素结合环、质子传递沟和位于K螺旋的绝对保守的"EXXR"模序。
CYP11B1基因产生突变后可引起11β-羟化酶不能表达或表达减少,从而导致11β-羟化酶缺陷症,并呈常染色体隐性遗传。目前,已报道的CYP11B1基因突变达70余种,其中有错义/无义突变、剪接突变、小片段缺失、大片段缺失插入突变及复杂重组(包括染色体倒位)。
随着科技进步,应用分子生物学及生物信息学技术,从分子水平对先天性肾上腺皮质增生症的发病机制和基因型——表型关系做进一步阐明,这必将是研究的新的切入点,亦为研究的重点,从而为其防治提供科学依据,以便更早地控制疾病的发生与发展,通过基因诊断及治疗,降低其发病率,延缓疾病进展。
研究目的：本研究通过对一个11β-羟化酶缺陷症家系中先证者及其家系成员CYP11B1基因的突变筛查,利用微小基因模型研究缺失突变的剪切机制,进而探索该11β-羟化酶缺陷症家系的分子遗传学机制。
研究方法：留取先证者及其他家系成员的外周血标本,提取基因组DNA；根据基因库中CYP11B1基因的序列,利用primer experss2.0软件设计引物,分别扩增该基因的9个外显子及其周围内含子序列。以家系成员的基因组DNA分别作为模板,进行PCR扩增,扩增产物纯化后应用美国ABI PRISM3700DNA自动测序仪进行测序,然后利用Autoassembler2.0软件将测序结果与UCSC数据库下载的基因组序列进行比较、分析,找出突变位点。由于发现的是大片段缺失突变,缺失片段包含外显子及内含子的5’剪切供体位点,将影响mRNA的剪接,因此我们进一步利用小基因模型(minigen)技术进行mRNA体外剪接研究,以明确该突变对11β-羟化酶的结构和功能造成的影响。首先,我们将包含CYP11B1基因的第3-6外显子的野生型及突变型片段构建到pcDNA3.1(+)质粒中,测序证实后转染293T细胞,提取RNA,以特异引物进行RT-PCR,扩增产物测序,并进行比对分析。
研究结果：1、家系资料：先证者李登科,男性,4岁6个月,因“身高增长过速2年半,乳腺及外生殖器发育1年半,加重半年”就诊。患者系足月顺产,头先露,出生时体重3.0kg,身长55cm,无抽搐,母乳喂养。患者于2年半之前出现身高增长过速,身高显著高于同龄其他儿童,且饮食量增加,智力较同龄儿童无明显差别。当时未行诊治。1年半之前出现乳腺及外生殖器发育,阴毛生长,阴茎可勃起,无遗精,期间身高一直在增加,遂到当地医院就诊,嘱暂予观察,未行其它特殊处理。半年前患者上述症状进一步加重,遂于半个月前赴济宁医学院附属医院检查,查血钾：2.60mmol/L,血清FT3:8.23pmol/L,雌二醇：55.05pg/ml,促黄体生成素：<0.100mlU/ml,促卵泡成熟激素：<0.100mIU/ml,未行特殊治疗,建议到上级医院做进一步检查。查体：T36.7℃,P84次／分,R19次／分,BP143/93mmHg, H134cm, W29.5kg,神志清,反应灵敏,会阴部皮肤颜色加深。无胡须,有喉结,两乳房发育。双肺呼吸音清,未闻及干湿性罗音。心率84次／分,律不齐,各瓣膜听诊区未闻及病理性杂音。腹部平坦,未见肠型及蠕动波,腹软、无压痛反跳痛,肝脾肋下未及,Murphy征阴性,肝区及肾区无叩击痛,肠鸣音正常。外生殖器发育,阴茎及双侧睾丸增大,无尿道下裂,外阴部阴毛生长。辅助检查示：血碱性磷酸酶365U／L,总胆固醇2.31mmol/L,钾2.7mmol/L,肝功生化其他项目在正常范围；甲功5项正常,β-HCG小于0.1mIU/ml, FSH0.23mIU/ml, LH小于0.1mIU/ml, E238.10mg/ml, PRG1.56ng/ml, TO1.14ng/ml, PRL9.94ng/ml,皮质醇66.98nmol/L(8点),71.15nmol/L(16点),ACTH1534pg/ml(8点),582.8pg/ml(16点)。X线片：腕骨二次骨化中心出现8块。尺、桡骨远端骨骺出现,但未闭合,掌指骨二次骨化中心出现,部分闭合,左膝骨骺未闭合。B超：前列腺发育,符合左肾上腺腺瘤声像图表现(左肾上腺低回声结节,约0.8×0.7×0.5cm)。最后诊断：先天性肾上腺皮质增生症,11-β羟化酶缺陷症并混合性性早熟。患者父母系近亲结婚,表型正常,患者有一姐姐,表型正常。
2、CYP11B1基因突变筛查结果：经过测序和序列比对分析,发现CYP11B1基因的第3-5外显子之间缺失449bp,患者为纯合缺失,而其父母、姐姐为杂合缺失。缺失的片段包括：第3外显子的3’端,全部内含子3和外显子4,第4内含子的5’剪切供体位点,其他外显子测序未发现突变位点。
3、致病机制研究结果：CYP11B1基因缺失突变(g.2697del449)将导致相应的mRNA缺失部分3号外显子和全部4号外显子,同时插入部分4号内含子序列,更重要的是,造成了从第167位氨基酸开始出现读码框改变,产生了大量错误的密码子,并在第217位氨基酸处产生了终止密码,造成翻译的CYP11B1蛋白出现提前终止,并完全破坏了该酶的血红素结合结构域,使该酶的活性完全丧失。
结论：通过对一个汉族11-β羟化酶缺陷症家系进行突变筛查,我们在国际上首次发现了CYP11B1基因上一个新的大片段缺失突变(g.2697del449),通过小基因系统模拟mRNA剪接,发现该缺失突变导致产生截断的蛋白,丧失了与血红素结合的结构域,造成11-β羟化酶功能完全丧失。CYP11B1基因缺失突变(g.2697del449)是该11-β羟化酶缺陷症家系的致病突变。
研究背景：促甲状腺激素(thyroid stimulating hormone, TSH)与促甲状腺激素受体(TSHR)是人体内参与甲状腺功能调控的两种重要蛋白分子。TSHR主要存在于甲状腺滤泡细胞膜上,生理情况下TSH与其结合后介导调节甲状腺滤泡细胞的正常生长和功能。近年来的研究证实,TSHR除存在于甲状腺滤泡细胞膜上外,尚存在于大脑、睾丸、肾脏、心肌、骨骼、胸腺、淋巴细胞、脂肪组织、纤维母细胞和肝细胞等多种甲状腺外组织和细胞上。TSH可能通过TSHR介导发挥着广泛的、非传统认识的功能作用,TSH的这种甲状腺外作用受到了广泛关注成为目前研究的热点。
甲状腺功能减退症(hypothyroidism,甲减)是“由于甲状腺激素缺乏或其生理效应不足所导致的机体代谢活动下降,而引起的一种内分泌疾病,实验室检查表现为TSH升高,甲状腺激素(FT3、FT4)降低”。甲状腺功能减退症常伴有血脂紊乱,易于出现动脉粥样硬化,增加心血管疾病的危险性。亚临床甲状腺功能减退症(subclinical hypothyroidism,亚甲减)亦是一种常见的内分泌专业亚临床疾病,主要诊断依据是血清TSH水平增高,而血清FT3、FT4正常[3]。亚临床甲减患病率较高,社区调查的患病率为3%～15%不等,女性、老年和高碘摄入量地区的患病率明显增加。美国Framingham队列研究报告：女性13%,男性5.7%；英国Whickham前瞻研究报告：女性7.5%,男性2.8%。亚临床甲减也常常伴有血脂紊乱,与动脉粥样硬化和缺血性心脏病密切相关。
既往人们一直认为：甲状腺功能减退症所伴随的血脂紊乱是由于甲状腺激素对血脂代谢的作用所致,但是,很显然,亚临床甲减所伴随的血脂紊乱不能用甲状腺激素的作用来解释。促甲状腺激素(TSH)在甲减及亚临床甲减患者均明显升高,但是其对血脂的影响目前研究较少。
冠状动脉粥样硬化性心脏病(Coronary heart disease),简称冠心病,是“因冠状动脉粥样硬化使血管腔狭窄或阻塞,或／和因冠状动脉功能性改变(痉挛)导致心肌缺血缺氧或坏死而引起的心脏病”。近年来,随着人口老龄化及心血管病危险因素的增加,冠心病的患病率和死亡率逐年升高,是全球死亡率最高的疾病之一,根据世界卫生组织2011年的报告,中国的冠心病死亡人数己列世界第二位。由国家心血管病中心编制的《中国心血管病报告2011》指出：“目前我国心血管病患者人数约为2.3亿,相当于每10个成年人中有2人患病。对于我国心血管病未来发展趋势,报告根据中国冠心病政策模型作出预测：2010年到2030年,如果仅考虑人口老龄化和人口增加的因素,中国35岁至84岁人群中心血管病事件数将增加50%以上；如果考虑血压、总胆固醇、糖尿病、吸烟的因素,心血管病事件数将额外增加23%。如果不改善应对措施,2005年至2015年,心血管疾病、卒中和糖尿病将会给中国造成5500亿美元的经济损失”。
冠心病的病因至今尚未完全清楚,但其己知的危险因素有许多,如高血压、血脂异常如高胆固醇血症和高低密度脂蛋白血症、糖尿病与胰岛素抵抗、年龄、吸烟、饮酒等,积极预防和治疗冠心病的危险因素,可降低冠心病患病率及死亡率。近年来,许多临床研究均显示甲状腺功能与冠心病关系密切,而且亚临床甲状腺功能减退能增加冠心病的发病率,是冠心病的重要危险因素之一。因此,对冠心病患者进行甲状腺功能状态分析对于预防和治疗冠心病有着重要意义。
我们前期基础研究证实：肝脏细胞表面存在TSHR, TSH与其结合促进肝脏合成胆固醇,在血胆固醇水平升高中发挥重要的作用。本研究通过对于冠心病患者甲状腺功能和血脂水平的分析,着重研究冠心病患者中TSH变化和血清胆固醇水平之间的关系,校正其他可能影响血脂的因素研究TSH和胆固醇之间关系,以期真实客观的反映TSH和与血脂之间的关系。
研究目的：探讨冠心病患者的TSH水平和血脂谱之间的关系。
研究方法：回顾性研究了1302例冠心病患者的资料,所有患者均经过冠状动脉造影确诊,并住院治疗。经过排除后,568例患者进入研究。首先对冠心病患者的甲状腺功能状态进行了分析,然后校正性别、年龄、血糖、吸烟状况及甲状腺激素水平等因素,应用Pearson's相关分析研究了TSH与血脂谱之间的关系。接下来,应用线性回归分析、通径分析、主成分回归分析及偏最小二乘法的方法分别研究了TSH水平对血胆固醇影响的程度。
研究结果：1、冠心病患者甲状腺功能状态分析显示：冠心病患者中,甲状腺功能异常的发生率是18.66%,其中甲状腺功能减退症(临床型和亚临床型)的发生率(15.32%)明显高于甲状腺功能亢进症的发生率(3.34%),女性甲功异常的发生率(26.17%)显著高于男性(10.37%)。
2、冠心病患者中,随着TSH水平的升高,高胆固醇的发生率呈线性增高的趋势(Pearson's Chi-squared test, linear trend0.010, p<0.05)。
3、当我们校正性别、年龄、吸烟状况、空腹血糖及甲状腺激素的影响后,冠心病患者的TSH水平与血TC水平依然呈显著正相关(r=0.095,p=0.036)。
4、在冠心病患者中排除性别年龄等因素的影响后,血清胆固醇随着TSH水平的升高而升高。偏最小二乘法显示：血清TSH每变化1各单位,血清胆固醇升高0.01558mmol/L
结论：TSH可以增加冠心病患者的血胆固醇水平。虽然TSH升高血胆固醇的作用较弱,但是仍然具有重要的生理作用及临床意义。当TSH水平升高,甚至在正常值高限时,就会增加高胆固醇血症的患病率。对冠心病患者来说,维持血TSH水平在合适的范围,将有助于控制血脂,减缓动脉粥样硬化的进展。
Background:Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders and is associated with significant morbidity and mortality rates in affected children and adults. Steroid11p-hydroxylase deficiency (110HD, OMIM:+202010), the second most common variant of CAH, accounts for approximately5-8%of CAH cases and occurs in1:100,000to1:200,000live births. Patients most commonly present with symptoms of androgen excess and significant hypertension, a hallmark of this CAH variant.11OHD is rarely described in China.
Inherited in an autosomally recessive manner,11OHD is caused by mutations in the CYP11B1gene (GeneID:1584; MIM:610613; GenBank:NC_000008.10), which is located on chromosome8q21, approximately40kb from the highly homologous aldosterone synthase gene (CYP11B2). Presently, over70CYP11B1-inactivating mutations are listed in the HGMD database (http://www.hgmd.org/). The majority of these mutations are missense and nonsense mutations; however, splice-site mutations, small deletions, small insertions, and complex rearrangements have also been detected.
Objective:To identify the mutation causing110HD in a Chinese pedigree and analyze the functional consequences and phenotype associated with this mutation.
Methods:A Chinese family with11OHD was screened for mutations in the CYP11B1gene. Genomic DNA was prepared from peripheral blood leukocytes sampled from the patient, his parents and sister using a standard proteinase K digestion and phenol-chloroform extraction. The whole CYP11B1gene, including all exons and introns, was directly amplified by polymerase chain reaction (PCR). Three pairs of primers were used to amplify exons1-2,3-5, and6-9of CYP11B1without amplifying the neighboring CYP11B2gene. The complete coding region of the CYP11B1gene, including the intron-exon boundaries, was directly sequenced using a dye-terminator cycle-sequencing system on an ABI Prism3700DNA sequencer (Applied Biosystems, Inc., Foster City, CA, USA). The resulting sequences were compared to the corresponding wild-type sequences of CYP11B1using the AutoAssembler software (version2.0; Perkin Elmer). Mini-gene experiment was performed to mimic the natural splicing and outcome of the genetic variation. A fragment containing the sequence from intron2to6of the CYP11B1gene were generated using the patient's and normal subject's genomic DNA as template with KOD-plus Polymerase (TOYOBO, Japan). The resulting products were ligated into the expression vector pcDNA3.1(Invitrogen, San Diego, CA, USA) between EcoRI and BamHI cloning sites. After confirmation of successful construction by direct DNA sequencing, four micrograms of normal or mutant plasmids were transfected into293T cells by the lipofectamine method (Lipo2000, Invitrogen, San Diego, CA, USA). RT-PCR was performed using the primers located in the exon3and exon5respectively. The PCR products was recovered from the gel and then sequenced with an ABI3730automated sequencer.
Results:The patient, a4.5-year-old Chinese boy, was first referred to our hospital for the treatment of rapid somatic growth, precocious virilization and mammoplasia. While there was no family or medical history of note, his parents were consanguineous. The patient's older sister was physically and mentally normal. The patient was born after an unremarkable pregnancy, during which no medication was taken, with a birth weight of3,000g and a birth length of55cm. The patient's growth rate accelerated2years ago; his voice deepened with the emergence of the laryngeal prominence. He also presented with axillary and pubic hair, facial acne, and reported erection of the penis. Breast development was also obvious. The patient's body height was134cm, which is+8.56standard deviations score (SDS) of normal controls with the same chromosomal sex and age, and his mean blood pressure was143/93mmHg, which is also above the normal range. The patient's weight was29.5kg. Physical examination showed hyperpigmentation of the skin of the perineum and mammary areola, a laryngeal prominence, and no facial hair. The patient was placed at Tanner stages P2B3G3, and his testicular volume was measured as2mL per testicle. The serum biochemical tests and hormone assays conducted prior to treatment showed that the patient had hypokalemia, a slightly lowered fasting blood glucose, highly increased ACTH with decreased cortisol, and dramatically increased levels of17-hydroxy-progesterone, testosterone and progesterone. Imaging examinations showed that the patient had a dramatically enlarged adrenal gland, a developed prostate, and an accelerated bone age (10yrs.)-Cytogenetic studies of the peripheral blood lymphocytes revealed a46XY karyotype.
Complete DNA sequencing of the CYP11B1gene revealed a novel449-bp homozygous deletion (g.2697de1449) in the patient and a heterozygous deletion in both of the patient's parents and sister. The deletion included the3'region of exon3, all of intron3and exon4, and the5'splice donor site of intron4.
This mutation was predicted to lead to the skipping of part of exon3and all of exon4in the CYP11B1mRNA and inserting of part of intron4. It generated a truncated protein and resulted in the complete destruction of the heme-binding domain of the enzyme.
Conclusion:The novel deletion drastically affects normal protein structure and abolishes normal enzyme activity, leading to a severe phenotype of congenital adrenal hyperplasia due to11OHD.
Background:The thyroid hormones exert a wide range of functions in several organs, including the heart. Abnormal thyroid hormone metabolism may lead to different forms of heart disease and hypothyroidism, in particular, is a well-known cause of accelerated coronary atherosclerosis. Moreover, similar consequences were found for subclinical hypothyroidism (SCH), which is characterized by elevated serum thyroid stimulating hormone (TSH) levels and normal thyroxine (T4) levels. Elevated TSH levels have recently aroused interest due to the potential for TSH to induce injury, especially in patients with coronary heart disease (CHD). A series of studies reported that a high level of TSH was associated with a deleterious change of serum lipids, with an increase of lipid abnormalities; however, this issue has been the subject of considerable debate, and several studies have not observed such an association. The differences in the studies have been ascribed to the influence of some confounding factors, such as age, gender and body mass index (BMI). Existing evidence has demonstrated that the relationship of TSH and lipid levels was different between overweight and normal weight populations and between men and women. Furthermore, the thyroid hormones play an important role in regulating lipid metabolism. Numerous studies have confirmed the presence of an inverse relationship between serum thyroxin and cholesterol levels. Even within the reference range, serum free thyroxine (FT4) levels near the upper limit have been associated with different metabolic markers in euthyroid subjects and patients with coronary artery disease. Therefore, to evaluate the essential relationship between TSH and the lipid status, it is necessary to adjust for age, gender, BMI and thyroid hormone levels. Regrettably, few studies have excluded the potential influences of the thyroid hormones when assessing the relationship between TSH and the lipid status.
Interestingly, in vivo and in vitro research by our laboratory on the function of TSH has shown that TSH, independent of thyroid hormones, can upregulate the expression of hepatic3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), which is the rate-limiting enzyme in cholesterol synthesis, and increase the cholesterol content in the liver. Therefore, we hypothesized that TSH, independent of thyroid hormones, would be positively associated with the serum cholesterol level.
The present study evaluated the relationship between TSH and the lipid status after adjusting for classic confounding factors and the thyroid hormones. We also analyzed the extent to which TSH can affect serum lipid parameters. The present study yielded insights into potentially novel effects of TSH on serum lipids and suggested that it is necessary to routinely test thyroid function in CHD patients. Maintaining serum TSH levels in an appropriate range will achieve homeostasis of the lipid levels and slow the progression of atherosclerosis in CHD patients.
Objective:The aim of the present study was to evaluate the relationship between serum TSH levels and the lipid profile independent of TH.
Methods:1302CHD patients diagnosed by coronary angiography were retrospectively studied. The prevalence and distribution of thyroid dysfunction were analyzed first. To assess the impact of TSH on serum lipids, Pearson's correlation analysis was performed after adjustments for classic factors and TH. To calculate the extent of the effect of TSH on the serum cholesterol level, the partial least squares method and additional statistical methods were used.
Results:After the exclusions, a total of568patients (270males and298females with a mean age of63.56±11.376years) were selected. The prevalence of thyroid dysfunction among the patients was18.66%, and the prevalence of hypothyroidism (15.32%) was higher than that of hyperthyroidism (3.34%). Even after adjusting for confounding factors, such as sex, age, smoking status, fasting plasma glucose levels and TH, a significant positive impact of TSH on the serum total cholesterol (TC) level was revealed (r=0.095, p=0.036). Each1mIU/L increase in the TSH level was linked to a0.015580712mmol/L elevation of the serum TC value.
Conclusion:TSH can increase the TC level in CHD patients independent of TH. The present study suggests a potential physiological role of TSH and the importance of maintaining an appropriate TSH level in CHD patients.
CAH是由于皮质类固醇激素合成过程中任何一种酶先天性缺陷,引起肾上腺皮质束状带合成的皮质醇完全或部分缺乏,经下丘脑-垂体-肾上腺轴反馈调节,引起促肾上腺皮质激素释放激素(CRH)和促肾上腺皮质激素(ACTH)分泌增加,导致了肾上腺皮质增生,另一方面,某一酶的缺陷也使得皮质醇生物合成链中缺陷酶近端的前体产物因不能继续向下游合成而堆积,和雄激素的过度生成。CAH临床主要特点为肾上腺皮质功能不全,性腺发育异常,伴或不伴水盐代谢紊乱,高血压。本症表现出女性男性化或性幼稚,男性假性性早熟或女性化,失盐或非失盐,高血压等一系列症状。
典型CAH的发病率约为1／10000,而非典型的发病率约为典型的10倍,因其临床表现错综复杂,且对疾病认识及实验室检查技术的不足,诊断方面尚存在许多困难,一般需要从临床表现,实验室检查和基因分析三方面进行综合诊断。目前CAH基因缺陷的相关检测技术没有推广,在我国临床工作中尚未常规开展。
11β-羟化酶缺陷症(steroid11β-hydroxylase deficiency,11-OHD, OMIM:+2020101是CAH中第二大类型,约占5-8%,发病率约为1／100,000至1／200,000。11β-羟化酶缺乏引起皮质醇合成减少,对ACTH的反馈抑制减弱,使下丘脑和垂体分泌的CRH及ACTH增加,导致束状带产生更多的皮质醇前体。这些前体物质通过旁路代谢,在网状带产生大量雄激素,导致高雄激素血症。此外,11-去氧皮质酮和11-去氧皮质醇水平升高,由于它们具有类盐皮质激素的作用,从而引起水钠潴留、高血压及低钾血症。11β-羟化酶缺陷症的临床表现包括经典型和非经典型。经典型11β-羟化酶缺陷症患者可表现为女性外生殖器男性化,男、女患者都可出现的假性性早熟,由于生长过快骨骺愈合提前引起身材矮小,以及血压升高、血钾降低。非经典型在女性患者多表现为月经紊乱,多毛和痤疮,类似多囊卵巢综合征的表现；在男性患者可无症状。
由于11β-羟化酶缺陷症的发病人数较少,其分子遗传学研究相对较少。现已证实：11β-羟化酶缺陷症的分子遗传学缺陷是由于CYP11B1基因突变所致。CYP11B1基因(GeneID:1584; MIM:610613; GenBank:NC_000008.10)位于第8号染色体长臂上的21区(8q21),由9个外显子组成(见图1-6),长度为6.03Kbp,编码503个氨基酸残基。
CYP11B1基因编码11β-羟化酶,该酶属于细胞色素P450蛋白超家族中的一员,在肾上腺束状带表达,位于线粒体内膜,催化11-去氧皮质醇和11-去氧皮质酮分别转变为皮质醇和皮质酮。11β-羟化酶分子量约47kDa,是一种含有血红素的蛋白,具有依赖还原型烟酰胺腺嘌呤二核苷酸磷酸(NAPDH)的氧化还原体系,该体系由黄素蛋白——皮质铁氧还蛋白还原酶及铁硫蛋白——皮质铁氧还蛋白构成。细胞色素P450蛋白家族的成员结构高度保守,其核心结构由一个被称作“曲”的螺旋,一个由J螺旋和K螺旋组成的四螺旋束以及两套β片层构成。该结构构成了血红素结合环、质子传递沟和位于K螺旋的绝对保守的"EXXR"模序。
CYP11B1基因产生突变后可引起11β-羟化酶不能表达或表达减少,从而导致11β-羟化酶缺陷症,并呈常染色体隐性遗传。目前,已报道的CYP11B1基因突变达70余种,其中有错义/无义突变、剪接突变、小片段缺失、大片段缺失插入突变及复杂重组(包括染色体倒位)。
随着科技进步,应用分子生物学及生物信息学技术,从分子水平对先天性肾上腺皮质增生症的发病机制和基因型——表型关系做进一步阐明,这必将是研究的新的切入点,亦为研究的重点,从而为其防治提供科学依据,以便更早地控制疾病的发生与发展,通过基因诊断及治疗,降低其发病率,延缓疾病进展。
研究目的：本研究通过对一个11β-羟化酶缺陷症家系中先证者及其家系成员CYP11B1基因的突变筛查,利用微小基因模型研究缺失突变的剪切机制,进而探索该11β-羟化酶缺陷症家系的分子遗传学机制。
研究方法：留取先证者及其他家系成员的外周血标本,提取基因组DNA；根据基因库中CYP11B1基因的序列,利用primer experss2.0软件设计引物,分别扩增该基因的9个外显子及其周围内含子序列。以家系成员的基因组DNA分别作为模板,进行PCR扩增,扩增产物纯化后应用美国ABI PRISM3700DNA自动测序仪进行测序,然后利用Autoassembler2.0软件将测序结果与UCSC数据库下载的基因组序列进行比较、分析,找出突变位点。由于发现的是大片段缺失突变,缺失片段包含外显子及内含子的5’剪切供体位点,将影响mRNA的剪接,因此我们进一步利用小基因模型(minigen)技术进行mRNA体外剪接研究,以明确该突变对11β-羟化酶的结构和功能造成的影响。首先,我们将包含CYP11B1基因的第3-6外显子的野生型及突变型片段构建到pcDNA3.1(+)质粒中,测序证实后转染293T细胞,提取RNA,以特异引物进行RT-PCR,扩增产物测序,并进行比对分析。
研究结果：1、家系资料：先证者李登科,男性,4岁6个月,因“身高增长过速2年半,乳腺及外生殖器发育1年半,加重半年”就诊。患者系足月顺产,头先露,出生时体重3.0kg,身长55cm,无抽搐,母乳喂养。患者于2年半之前出现身高增长过速,身高显著高于同龄其他儿童,且饮食量增加,智力较同龄儿童无明显差别。当时未行诊治。1年半之前出现乳腺及外生殖器发育,阴毛生长,阴茎可勃起,无遗精,期间身高一直在增加,遂到当地医院就诊,嘱暂予观察,未行其它特殊处理。半年前患者上述症状进一步加重,遂于半个月前赴济宁医学院附属医院检查,查血钾：2.60mmol/L,血清FT3:8.23pmol/L,雌二醇：55.05pg/ml,促黄体生成素：<0.100mlU/ml,促卵泡成熟激素：<0.100mIU/ml,未行特殊治疗,建议到上级医院做进一步检查。查体：T36.7℃,P84次／分,R19次／分,BP143/93mmHg, H134cm, W29.5kg,神志清,反应灵敏,会阴部皮肤颜色加深。无胡须,有喉结,两乳房发育。双肺呼吸音清,未闻及干湿性罗音。心率84次／分,律不齐,各瓣膜听诊区未闻及病理性杂音。腹部平坦,未见肠型及蠕动波,腹软、无压痛反跳痛,肝脾肋下未及,Murphy征阴性,肝区及肾区无叩击痛,肠鸣音正常。外生殖器发育,阴茎及双侧睾丸增大,无尿道下裂,外阴部阴毛生长。辅助检查示：血碱性磷酸酶365U／L,总胆固醇2.31mmol/L,钾2.7mmol/L,肝功生化其他项目在正常范围；甲功5项正常,β-HCG小于0.1mIU/ml, FSH0.23mIU/ml, LH小于0.1mIU/ml, E238.10mg/ml, PRG1.56ng/ml, TO1.14ng/ml, PRL9.94ng/ml,皮质醇66.98nmol/L(8点),71.15nmol/L(16点),ACTH1534pg/ml(8点),582.8pg/ml(16点)。X线片：腕骨二次骨化中心出现8块。尺、桡骨远端骨骺出现,但未闭合,掌指骨二次骨化中心出现,部分闭合,左膝骨骺未闭合。B超：前列腺发育,符合左肾上腺腺瘤声像图表现(左肾上腺低回声结节,约0.8×0.7×0.5cm)。最后诊断：先天性肾上腺皮质增生症,11-β羟化酶缺陷症并混合性性早熟。患者父母系近亲结婚,表型正常,患者有一姐姐,表型正常。
2、CYP11B1基因突变筛查结果：经过测序和序列比对分析,发现CYP11B1基因的第3-5外显子之间缺失449bp,患者为纯合缺失,而其父母、姐姐为杂合缺失。缺失的片段包括：第3外显子的3’端,全部内含子3和外显子4,第4内含子的5’剪切供体位点,其他外显子测序未发现突变位点。
3、致病机制研究结果：CYP11B1基因缺失突变(g.2697del449)将导致相应的mRNA缺失部分3号外显子和全部4号外显子,同时插入部分4号内含子序列,更重要的是,造成了从第167位氨基酸开始出现读码框改变,产生了大量错误的密码子,并在第217位氨基酸处产生了终止密码,造成翻译的CYP11B1蛋白出现提前终止,并完全破坏了该酶的血红素结合结构域,使该酶的活性完全丧失。
结论：通过对一个汉族11-β羟化酶缺陷症家系进行突变筛查,我们在国际上首次发现了CYP11B1基因上一个新的大片段缺失突变(g.2697del449),通过小基因系统模拟mRNA剪接,发现该缺失突变导致产生截断的蛋白,丧失了与血红素结合的结构域,造成11-β羟化酶功能完全丧失。CYP11B1基因缺失突变(g.2697del449)是该11-β羟化酶缺陷症家系的致病突变。
研究背景：促甲状腺激素(thyroid stimulating hormone, TSH)与促甲状腺激素受体(TSHR)是人体内参与甲状腺功能调控的两种重要蛋白分子。TSHR主要存在于甲状腺滤泡细胞膜上,生理情况下TSH与其结合后介导调节甲状腺滤泡细胞的正常生长和功能。近年来的研究证实,TSHR除存在于甲状腺滤泡细胞膜上外,尚存在于大脑、睾丸、肾脏、心肌、骨骼、胸腺、淋巴细胞、脂肪组织、纤维母细胞和肝细胞等多种甲状腺外组织和细胞上。TSH可能通过TSHR介导发挥着广泛的、非传统认识的功能作用,TSH的这种甲状腺外作用受到了广泛关注成为目前研究的热点。
甲状腺功能减退症(hypothyroidism,甲减)是“由于甲状腺激素缺乏或其生理效应不足所导致的机体代谢活动下降,而引起的一种内分泌疾病,实验室检查表现为TSH升高,甲状腺激素(FT3、FT4)降低”。甲状腺功能减退症常伴有血脂紊乱,易于出现动脉粥样硬化,增加心血管疾病的危险性。亚临床甲状腺功能减退症(subclinical hypothyroidism,亚甲减)亦是一种常见的内分泌专业亚临床疾病,主要诊断依据是血清TSH水平增高,而血清FT3、FT4正常[3]。亚临床甲减患病率较高,社区调查的患病率为3%～15%不等,女性、老年和高碘摄入量地区的患病率明显增加。美国Framingham队列研究报告：女性13%,男性5.7%；英国Whickham前瞻研究报告：女性7.5%,男性2.8%。亚临床甲减也常常伴有血脂紊乱,与动脉粥样硬化和缺血性心脏病密切相关。
既往人们一直认为：甲状腺功能减退症所伴随的血脂紊乱是由于甲状腺激素对血脂代谢的作用所致,但是,很显然,亚临床甲减所伴随的血脂紊乱不能用甲状腺激素的作用来解释。促甲状腺激素(TSH)在甲减及亚临床甲减患者均明显升高,但是其对血脂的影响目前研究较少。
冠状动脉粥样硬化性心脏病(Coronary heart disease),简称冠心病,是“因冠状动脉粥样硬化使血管腔狭窄或阻塞,或／和因冠状动脉功能性改变(痉挛)导致心肌缺血缺氧或坏死而引起的心脏病”。近年来,随着人口老龄化及心血管病危险因素的增加,冠心病的患病率和死亡率逐年升高,是全球死亡率最高的疾病之一,根据世界卫生组织2011年的报告,中国的冠心病死亡人数己列世界第二位。由国家心血管病中心编制的《中国心血管病报告2011》指出：“目前我国心血管病患者人数约为2.3亿,相当于每10个成年人中有2人患病。对于我国心血管病未来发展趋势,报告根据中国冠心病政策模型作出预测：2010年到2030年,如果仅考虑人口老龄化和人口增加的因素,中国35岁至84岁人群中心血管病事件数将增加50%以上；如果考虑血压、总胆固醇、糖尿病、吸烟的因素,心血管病事件数将额外增加23%。如果不改善应对措施,2005年至2015年,心血管疾病、卒中和糖尿病将会给中国造成5500亿美元的经济损失”。
冠心病的病因至今尚未完全清楚,但其己知的危险因素有许多,如高血压、血脂异常如高胆固醇血症和高低密度脂蛋白血症、糖尿病与胰岛素抵抗、年龄、吸烟、饮酒等,积极预防和治疗冠心病的危险因素,可降低冠心病患病率及死亡率。近年来,许多临床研究均显示甲状腺功能与冠心病关系密切,而且亚临床甲状腺功能减退能增加冠心病的发病率,是冠心病的重要危险因素之一。因此,对冠心病患者进行甲状腺功能状态分析对于预防和治疗冠心病有着重要意义。
我们前期基础研究证实：肝脏细胞表面存在TSHR, TSH与其结合促进肝脏合成胆固醇,在血胆固醇水平升高中发挥重要的作用。本研究通过对于冠心病患者甲状腺功能和血脂水平的分析,着重研究冠心病患者中TSH变化和血清胆固醇水平之间的关系,校正其他可能影响血脂的因素研究TSH和胆固醇之间关系,以期真实客观的反映TSH和与血脂之间的关系。
研究目的：探讨冠心病患者的TSH水平和血脂谱之间的关系。
研究方法：回顾性研究了1302例冠心病患者的资料,所有患者均经过冠状动脉造影确诊,并住院治疗。经过排除后,568例患者进入研究。首先对冠心病患者的甲状腺功能状态进行了分析,然后校正性别、年龄、血糖、吸烟状况及甲状腺激素水平等因素,应用Pearson's相关分析研究了TSH与血脂谱之间的关系。接下来,应用线性回归分析、通径分析、主成分回归分析及偏最小二乘法的方法分别研究了TSH水平对血胆固醇影响的程度。
研究结果：1、冠心病患者甲状腺功能状态分析显示：冠心病患者中,甲状腺功能异常的发生率是18.66%,其中甲状腺功能减退症(临床型和亚临床型)的发生率(15.32%)明显高于甲状腺功能亢进症的发生率(3.34%),女性甲功异常的发生率(26.17%)显著高于男性(10.37%)。
2、冠心病患者中,随着TSH水平的升高,高胆固醇的发生率呈线性增高的趋势(Pearson's Chi-squared test, linear trend0.010, p<0.05)。
3、当我们校正性别、年龄、吸烟状况、空腹血糖及甲状腺激素的影响后,冠心病患者的TSH水平与血TC水平依然呈显著正相关(r=0.095,p=0.036)。
4、在冠心病患者中排除性别年龄等因素的影响后,血清胆固醇随着TSH水平的升高而升高。偏最小二乘法显示：血清TSH每变化1各单位,血清胆固醇升高0.01558mmol/L
结论：TSH可以增加冠心病患者的血胆固醇水平。虽然TSH升高血胆固醇的作用较弱,但是仍然具有重要的生理作用及临床意义。当TSH水平升高,甚至在正常值高限时,就会增加高胆固醇血症的患病率。对冠心病患者来说,维持血TSH水平在合适的范围,将有助于控制血脂,减缓动脉粥样硬化的进展。
Background:Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders and is associated with significant morbidity and mortality rates in affected children and adults. Steroid11p-hydroxylase deficiency (110HD, OMIM:+202010), the second most common variant of CAH, accounts for approximately5-8%of CAH cases and occurs in1:100,000to1:200,000live births. Patients most commonly present with symptoms of androgen excess and significant hypertension, a hallmark of this CAH variant.11OHD is rarely described in China.
Inherited in an autosomally recessive manner,11OHD is caused by mutations in the CYP11B1gene (GeneID:1584; MIM:610613; GenBank:NC_000008.10), which is located on chromosome8q21, approximately40kb from the highly homologous aldosterone synthase gene (CYP11B2). Presently, over70CYP11B1-inactivating mutations are listed in the HGMD database (http://www.hgmd.org/). The majority of these mutations are missense and nonsense mutations; however, splice-site mutations, small deletions, small insertions, and complex rearrangements have also been detected.
Objective:To identify the mutation causing110HD in a Chinese pedigree and analyze the functional consequences and phenotype associated with this mutation.
Methods:A Chinese family with11OHD was screened for mutations in the CYP11B1gene. Genomic DNA was prepared from peripheral blood leukocytes sampled from the patient, his parents and sister using a standard proteinase K digestion and phenol-chloroform extraction. The whole CYP11B1gene, including all exons and introns, was directly amplified by polymerase chain reaction (PCR). Three pairs of primers were used to amplify exons1-2,3-5, and6-9of CYP11B1without amplifying the neighboring CYP11B2gene. The complete coding region of the CYP11B1gene, including the intron-exon boundaries, was directly sequenced using a dye-terminator cycle-sequencing system on an ABI Prism3700DNA sequencer (Applied Biosystems, Inc., Foster City, CA, USA). The resulting sequences were compared to the corresponding wild-type sequences of CYP11B1using the AutoAssembler software (version2.0; Perkin Elmer). Mini-gene experiment was performed to mimic the natural splicing and outcome of the genetic variation. A fragment containing the sequence from intron2to6of the CYP11B1gene were generated using the patient's and normal subject's genomic DNA as template with KOD-plus Polymerase (TOYOBO, Japan). The resulting products were ligated into the expression vector pcDNA3.1(Invitrogen, San Diego, CA, USA) between EcoRI and BamHI cloning sites. After confirmation of successful construction by direct DNA sequencing, four micrograms of normal or mutant plasmids were transfected into293T cells by the lipofectamine method (Lipo2000, Invitrogen, San Diego, CA, USA). RT-PCR was performed using the primers located in the exon3and exon5respectively. The PCR products was recovered from the gel and then sequenced with an ABI3730automated sequencer.
Results:The patient, a4.5-year-old Chinese boy, was first referred to our hospital for the treatment of rapid somatic growth, precocious virilization and mammoplasia. While there was no family or medical history of note, his parents were consanguineous. The patient's older sister was physically and mentally normal. The patient was born after an unremarkable pregnancy, during which no medication was taken, with a birth weight of3,000g and a birth length of55cm. The patient's growth rate accelerated2years ago; his voice deepened with the emergence of the laryngeal prominence. He also presented with axillary and pubic hair, facial acne, and reported erection of the penis. Breast development was also obvious. The patient's body height was134cm, which is+8.56standard deviations score (SDS) of normal controls with the same chromosomal sex and age, and his mean blood pressure was143/93mmHg, which is also above the normal range. The patient's weight was29.5kg. Physical examination showed hyperpigmentation of the skin of the perineum and mammary areola, a laryngeal prominence, and no facial hair. The patient was placed at Tanner stages P2B3G3, and his testicular volume was measured as2mL per testicle. The serum biochemical tests and hormone assays conducted prior to treatment showed that the patient had hypokalemia, a slightly lowered fasting blood glucose, highly increased ACTH with decreased cortisol, and dramatically increased levels of17-hydroxy-progesterone, testosterone and progesterone. Imaging examinations showed that the patient had a dramatically enlarged adrenal gland, a developed prostate, and an accelerated bone age (10yrs.)-Cytogenetic studies of the peripheral blood lymphocytes revealed a46XY karyotype.
Complete DNA sequencing of the CYP11B1gene revealed a novel449-bp homozygous deletion (g.2697de1449) in the patient and a heterozygous deletion in both of the patient's parents and sister. The deletion included the3'region of exon3, all of intron3and exon4, and the5'splice donor site of intron4.
This mutation was predicted to lead to the skipping of part of exon3and all of exon4in the CYP11B1mRNA and inserting of part of intron4. It generated a truncated protein and resulted in the complete destruction of the heme-binding domain of the enzyme.
Conclusion:The novel deletion drastically affects normal protein structure and abolishes normal enzyme activity, leading to a severe phenotype of congenital adrenal hyperplasia due to11OHD.
Background:The thyroid hormones exert a wide range of functions in several organs, including the heart. Abnormal thyroid hormone metabolism may lead to different forms of heart disease and hypothyroidism, in particular, is a well-known cause of accelerated coronary atherosclerosis. Moreover, similar consequences were found for subclinical hypothyroidism (SCH), which is characterized by elevated serum thyroid stimulating hormone (TSH) levels and normal thyroxine (T4) levels. Elevated TSH levels have recently aroused interest due to the potential for TSH to induce injury, especially in patients with coronary heart disease (CHD). A series of studies reported that a high level of TSH was associated with a deleterious change of serum lipids, with an increase of lipid abnormalities; however, this issue has been the subject of considerable debate, and several studies have not observed such an association. The differences in the studies have been ascribed to the influence of some confounding factors, such as age, gender and body mass index (BMI). Existing evidence has demonstrated that the relationship of TSH and lipid levels was different between overweight and normal weight populations and between men and women. Furthermore, the thyroid hormones play an important role in regulating lipid metabolism. Numerous studies have confirmed the presence of an inverse relationship between serum thyroxin and cholesterol levels. Even within the reference range, serum free thyroxine (FT4) levels near the upper limit have been associated with different metabolic markers in euthyroid subjects and patients with coronary artery disease. Therefore, to evaluate the essential relationship between TSH and the lipid status, it is necessary to adjust for age, gender, BMI and thyroid hormone levels. Regrettably, few studies have excluded the potential influences of the thyroid hormones when assessing the relationship between TSH and the lipid status.
Interestingly, in vivo and in vitro research by our laboratory on the function of TSH has shown that TSH, independent of thyroid hormones, can upregulate the expression of hepatic3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), which is the rate-limiting enzyme in cholesterol synthesis, and increase the cholesterol content in the liver. Therefore, we hypothesized that TSH, independent of thyroid hormones, would be positively associated with the serum cholesterol level.
The present study evaluated the relationship between TSH and the lipid status after adjusting for classic confounding factors and the thyroid hormones. We also analyzed the extent to which TSH can affect serum lipid parameters. The present study yielded insights into potentially novel effects of TSH on serum lipids and suggested that it is necessary to routinely test thyroid function in CHD patients. Maintaining serum TSH levels in an appropriate range will achieve homeostasis of the lipid levels and slow the progression of atherosclerosis in CHD patients.
Objective:The aim of the present study was to evaluate the relationship between serum TSH levels and the lipid profile independent of TH.
Methods:1302CHD patients diagnosed by coronary angiography were retrospectively studied. The prevalence and distribution of thyroid dysfunction were analyzed first. To assess the impact of TSH on serum lipids, Pearson's correlation analysis was performed after adjustments for classic factors and TH. To calculate the extent of the effect of TSH on the serum cholesterol level, the partial least squares method and additional statistical methods were used.
Results:After the exclusions, a total of568patients (270males and298females with a mean age of63.56±11.376years) were selected. The prevalence of thyroid dysfunction among the patients was18.66%, and the prevalence of hypothyroidism (15.32%) was higher than that of hyperthyroidism (3.34%). Even after adjusting for confounding factors, such as sex, age, smoking status, fasting plasma glucose levels and TH, a significant positive impact of TSH on the serum total cholesterol (TC) level was revealed (r=0.095, p=0.036). Each1mIU/L increase in the TSH level was linked to a0.015580712mmol/L elevation of the serum TC value.
Conclusion:TSH can increase the TC level in CHD patients independent of TH. The present study suggests a potential physiological role of TSH and the importance of maintaining an appropriate TSH level in CHD patients.
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46. 赵家军,垂体激素与靶器官外组织的对话-TSH在甲状腺外存在广泛的作用，in中华医学会第十次全国内分泌学学术会议论文汇编.2011：中国苏州.
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28. Rodondi N, A.D., Vittinghoff E,et al., Subclinical hypothyroidism and the risk of coronary heart disease:a meta-analysis. The American Journal of Medicine, 2006.119(7):p.541-551.
29. LH., D., Thyroid disease and lipids. Thyroid,2002.12:p.287-293.
30. Hak, A.E., Pols, H.A., Visser, T.J., Drexhage, H.A., Hofman, A., and Witteman, J.C., Subclinical hypothyroidism is an independent risk factor for atherosclerosis and myocardial infarction in elderly women:the Rotterdam Study. Ann Intern Med,2000.132:p.270-278.
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32. Asvold BO, V.L., Nilsen TI, BJoro T., The association between TSH within the reference range and serum lipid concentrations in a population-based study. The HUNT Study. Eur J Endocrinol,2007.156(2):p.181-186.
33. Canaris GJ, M.N., Mayor G, Ridgway EC., The Colorado thyroid disease prevalence study. Arch Intern Med,2000160(4):p.526-534.
34. Park SB, C.H., Joo NS., J Korean Med Sci. The relation of thyroid function to components of the metabolic syndrome in Korean men and women.,2011. 26(4):p.540-545.
35. Lu L, W.B., Shan Z, Jiang F, Teng X, Chen Y, Lai Y, Wang J, Xue H, Wang S, Li C, Liu H, Li N, Yu J, Shi L, Hou X, Xing Q, Bai X, Teng W., The correlation between thyrotropin and dyslipidemia in a population-based study. J Korean Med Sci,2011.26(2):p.243-249.
36. Fernandez-Real JM, L.-B.A., Castro A, Casamitjana R, Ricart W., Thyroid function is intrinsically linked to insulin sensitivity and endothelium-dependent vasodilation in healthy euthyroid subjects. J Clin Endocrinol Metab.,2006 91(9):p.3337-3343.
37. Garduno-Garcia Jde J, A.-G.U., Lopez-Carrasco G, Padilla Mendoza ME, Mehta R, Arellano-Campos O, Choza R, Sauque L, Garay-Sevilla ME, Malacara JM, Gomez-Perez FJ, Aguilar-Salinas CA., TSH and free thyroxine concentrations are associated with differing metabolic markers in euthyroid subjects. Eur J Endocrinol.,2010.163(2):p.273-278.
38. Vierhapper H, N.A., Grosser P, Raber W, Gessl A., Low-density lipoprotein cholesterol in subclinical hypothyroidism. Thyroid,2000.10:p.981-984.
39. Tunbridge WM, E.D., Hall R, Appleton D, Brewis M, Clark F, Evans JG, Young E, Bird T, Smith PA., Lipid profiles and cardiovascular disease in the Whickham area with particular reference to thyroid failure. Clin Endocrinol (Oxf).1977.7(6):p.495-508.
40. Hollowell JG, S.N., Flanders WD, Hannon WH, Gunter EW, Spencer CA, Braverman LE., Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994):National Health and Nutrition Examination Survey (NHANESIII). J Clin Endocrinol Metab.,2002.87(2):p.489-99.
41. Hueston WJ, P.W., Subclinical hypothyroidism and the risk of hypercholesterolemia. Ann Fam Med.,2004.2(4):p.351-355.
42. LH., D., Thyroid disease and lipids. Thyroid.,2002.12(4):p.287-293.
43. Maugeri D, S.A., Barbagallo P, Bonanno MR, Malaguarnera M, Rizza I, Speciale S, Tomarchio M, Curasi MP, Panebianco P., Thyroid hormones and lipid metabolism in a group of patients over seventy. Eur Rev Med Pharmacol Sci.,1999.3(5):p.211-216.
44. Bindels AJ, W.R., Frolich M, Seidell JC, Blokstra A, Smelt AH., The prevalence of subclinical hypothyroidism at different total plasma cholesterol levels in middle aged men and women:a need for case-finding? Clin Endocrinol (Oxt).1999.50(2):p.217-20.
45. Waterhouse DF, M.A., Walsh CD, Sheehan F, O'Shea D., An examination of the relationship between normal range thyrotropin and cardiovascular risk parameters:a study in healthy women. Thyroid.,2007.17(3).
46. 赵家军,垂体激素与靶器官外组织的对话-TSH在甲状腺外存在广泛的作用，in中华医学会第十次全国内分泌学学术会议论文汇编.2011：中国苏州.
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