Identification And Functional Characterization Of A Large Deletion Of The CYP11B1Gene Causing An11β-hydroxylase Deficiency In A Chinese Pedigree Thyroid Stimulating Hormone, Independent Of Thyroid Hormone,Can Elevate The Serum Total Cholesterol Level In

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:NC₀00008.10）, 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.