Chung Choo Lee

Two partial deletion mutations involving the same Alu sequence within intron 8 of the LDL receptor gene in Korean patients with familial hypercholesterolemia

Abstract Twenty-eight unrelated persons heterozygous for familial hypercholesterolemia (FH) were screened to assess the frequency and nature of major structural rearrangements at the low-density lipoprotein (LDL) receptor gene in Korean FH patients. Genomic DNA was analyzed by Southern blot hybridization with probes encompassing exons 1–18 of the LDL receptor gene. Two different deletion mutations (FH29 and FH110) were detected in three FH patients (10.7%). Each of the mutations was characterized by the use of exon-specific probes and detailed restriction mapping mediated by long-PCR (polymerase chain reaction). Mutation FH29 was a 3.83-kb deletion extending from intron 6 to intron 8 and FH110 was a 5.71-kb deletion extending from intron 8 to intron 12. In FH29, the translational reading frame was preserved and the deducible result was a cysteine-rich A and B repeat truncated protein that might be unable to bind LDL but would continue to bind β-VLDL. FH110 is presumed to be a null allele, since the deletion shifts the reading frame and results in a truncated protein that terminates in exon 13. Sequence analysis revealed that both deletions have occurred between two Alu-repetitive sequences that are in the same orientation. This suggested that in these patients the deletions were caused by an unequal crossing over event following mispairing of two Alu sequences on different chromatids during meiosis. Moreover, in both deletions, the recombinations were related to an Alu sequence in intron 8 and the deletion breakpoints are found within a specific sequence, 27 bp in length. This supports the hypothesis that this region might have some intrinsic instability, and act as one of the important factors in large recombinational rearrangements.

NMDA Receptor Antagonist Decreases the Progesterone-Induced Increase in GnRH Gene Expression in the Rat Hypothalamus

We previously reported that GnRH gene expression was enhanced by progesterone (P) in the hypothalamus from ovariectomized and estrogen (OVX+E) treated immature rats. Recent studies indicate that excitatory amino acids may play an important role in the regulation of GnRH secretion and gene expression by steroids. Therefore the present study attempts to examine whether excitatory amino acids are involved in the P-induced GnRH gene expression and release in vitro. MK-801, an NMDA receptor antagonist or CNQX, a non-NMDA receptor antagonist, was administered to OVX+E+P-treated prepubertal female rats. GnRH mRNA was determined by Northern blot hybridization using 32P-labeled antisense RNA, and GnRH release in vitro from the hypothalamic fragments was monitored by GnRH radioimmunoassay. The administration of MK-801 (0.2 mg/kg) for 2 h significantly reduced the P-induced GnRH gene expression and release, whereas CNQX (0.4 mg/kg) had no effect. These results clearly indicate that excitatory amino acids by way of NMDA receptor are involved in the transsynaptic regulation of GnRH gene expression.

A Partial Blockade of Catecholaminergic Neurotransmission with 6-Hydroxydopamine Decreases mRNA Level of Gonadotropin Releasing Hormone in the Male Rat Hypothalamus

Central catecholamines (CA) are known to be involved in the regulation of synthesis and secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus. However, no attempt has been yet made to determine whether CA affects GnRH gene expression. To this end, the effect of 6-hydroxydopamine (6-OHDA), a catecholaminergic neurotoxin, on GnRH mRNA level was examined. Hypothalamic tissues obtained from adult male rats were incubated with medium containing 6-OHDA. To ensure the effect of 6-OHDA on CA depleting action, CA levels in media and in postincubation tissues were determined. Increasing concentrations of 6-OHDA resulted in decrease in norepinephrine (NE) and dopamine (DA) contents in a dose dependent manner. Treatment with 6-OHDA (5 x 10(-4) M produced a time-dependent decrease in NE but not DA, when CA levels in media were determined at 30 min intervals during the incubation period. To determine changes in GnRH mRNA level in response to 6-OHDA treatment in vitro, for 2.5 h total cytoplasmic RNA fractions were isolated from postincubation hypothalamic tissues and used for RNA-blot hybridization with 32P-labeled GnRH riboprobe. A blockade of CA neurotransmission with 6-OHDA (5 x 10(-4) M) significantly reduced GnRH mRNA level by half over its control and internal control (actin mRNA) groups. Northern blot analysis revealed that addition of NE (1 x 10(-6) M) reversed the decreased GnRH mRNA level by 6-OHDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Red cell enzyme and serum protein polymorphisms in South Korea.

Two population groups in South Korea, one from Kwangju and one from Kangreung, were studied in regard to the erythrocyte enzyme polymorphisms GPT, ACP, GLO, ESD, 6PGD, ADA, AK, PGP and subtypes of PGM1 as well as regarding the serum protein variants of C3, HP, BF, PLG, AMY and the subtypes of GC, TF and PI. The results were compared with data of the population groups from the area of Cheju Island, Taejon and Seoul. The Korean population showed a rather high degree of genetic homogeneity.

Genetic variation of the angiotensin-converting enzyme gene: increased frequency of the insertion allele in Koreans

In view of the clinical importance of angiotensin‐converting enzyme (ACE) as a major marker for cardiovascular diseases, we investigated insertion/deletion (I/D) polymorphism of the ACE gene in Koreans. Genotype frequencies were examined by polymerase chain reaction in 171 patients with coronary artery disease (CAD) and 120 healthy subjects. Allele frequencies of ACE polymorphism in Koreans were not significantly different between patient and control groups. In addition, association between ACE genotypes and the number of stenosed coronary arteries was not detected. ACE genotypes in the CAD group were not associated with body mass index and plasma lipid levels. Thus, our results suggest that, at least in Koreans, I/D polymorphism of the gene is unlikely to be a useful marker for CAD subjects. However, the I allele frequency of Koreans (0.58) was higher than that of Caucasian populations (0.47) but lower than that of Samoan (0.91) and Yanomami (0.85) populations. Here, we discuss the clinical and ethnic importance of ACE polymorphism.

Evidence for multiple forms of the prothoracicotropic hormone in Drosophila melanogaster and indication of a new function

Abstract The prothoracicotropic hormone (PTTH) was extracted from brains of the fruit fly, Drosophila melanogaster, partially purified and characterized by chromatography and electrophoresis. In vitro bioassays of the fractions obtained from chromatography revealed the existence of at least two forms of PTTH, a large form (14–17 kDa: “big PTTH”) and a small form (3–5 kDa: “small PTTH”). The neural extract was able to stimulate ring glands to secrete ecdysone in a dose-dependent manner in vitro. Moreover, when incubated with imaginal wing discs, the extract specifically stimulated the synthesis of the heat-shock protein 83. This observation suggests that PTTH may have a hormonal role beyond its well-known function in the control of ecdysone secretion within the neuro-endocrine axis of brain and prothoracic glands.

Noradrenergic Neurotoxin Suppresses Gonadotropin‐Releasing Hormone (GnRH) and GnRH Receptor Gene Expression in Ovariectomized and Steroid‐Treated Rats

The present study was designed to investigate whether noradrenergic neurotransmission regulates the gene expression of gonadotropin‐releasing hormone (GnRH) in the preoptic area and GnRH receptor in the pituitary. To this end, N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP4, 50 mg/kg), an intraperitoneal (i.p.) injection of selective noradrenergic neurotoxin, was administered 1 h before progesterone (1 mg) treatment in ovariectomized and estradiol‐treated prepubertal rats. Treatment with DSP4 effectively blocked the progesterone‐induced increase in hypothalamic noradrenaline content, but not dopamine content, indicating that DSP4 selectively inhibits noradrenergic neurotransmission. DSP4 significantly blocked progesterone‐induced increase in serum luteinizing hormone (LH) concentrations as well as GnRH release from hypothalamic fragments incubated in vitro. DSP4 concomitantly down‐regulated GnRH mRNA levels in the preoptic area, as determined by competitive reverse transcription‐polymerase chain reaction. DSP4 also clearly down‐regulated progesterone‐induced GnRH receptor mRNA levels in the pituitary, whereas it failed to alter LHβ mRNA levels. In summary, blockade of noradrenergic neurotransmission with DSP4 resulted in profound reductions of hypothalamic GnRH and pituitary GnRH receptor gene expression.

Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men

OBJECTIVE To investigate whether or not alternatively spliced variants of the FSH receptor gene occur in human testis and whether the presence of the splicing variants is associated with spermatogenic defects and serum FSH concentration in infertile men. DESIGN A prospective case control study. SETTING An IVF clinic and infertility laboratory at a university hospital. PATIENT(S) Forty-three infertile patients undergoing testicular biopsy. INTERVENTION(S) Total RNA was extracted from the testicular tissues and used for reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S) Expression pattern was analyzed by nested RT-PCR using primers designed to amplify a fragment of FSH receptor gene. PCR products of splicing variants were cloned and sequenced. RESULT(S) The PCR products showed three kinds of additional bands corresponding to alternatively spliced isoforms of the FSH receptor gene. Exon 9 deleted variant was detected in all patients and inclusion variant of small extra exon was detected in 64% (9/14) of the patients with normal spermatogenesis and 34% (10/29) of the patients with spermatogenic defects. The presence of inclusion variant was not significantly associated with spermatogenic defects but was associated with a low level of serum FSH. On the other hand, exon 6 deleted variant was detected in only one patient having a high level of FSH concentration (30 IU/L) and Sertoli cell only syndrome. CONCLUSION(S) We identified three different types of alternatively spliced variants of the human FSH receptor. However, it is not clear whether or not there is an association between three variants and spermatogenic defects.

Identification of four novel mutations of the low‐density lipoprotein receptor gene in Korean patients with familial hypercholesterolemia

To obtain insight into the genetic variation of the low‐density lipoprotein (LDL) receptor gene in Korean patients with familial hypercholesterolemia (FH), we used single‐strand conformation polymorphism to screen all 18 exons and a promotor of the LDL receptor gene in 20 unrelated Korean FH patients. Four novel point mutations were detected in 5 FH patients and were characterized by sequence analysis. Of them, one is a nonsense mutation, a Glu→Stop (C AG→T AG) at codon 161, and results in a large deletion. The other three, which were a Ala→Glu (GC G→GĀG) mutation at signal peptide, Cys→Tyr (TG C→TĀC) at codon 210, and Pro→Leu (CT G→CC G) at codon 584, were novel missense mutations, which modified the highly conserved region of the LDL receptor gene. All these mutations were absent in normolipidemic controls and were associated in heterozygote carriers with clinical signs of FH. Identification of these novel mutations provides another example of the molecular heterogeneity of the LDL receptor gene mutations causing FH.

Three novel small deletion mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia

The low‐density lipoprotein (LDL) receptor gene from 80 unrelated Korean patients with familial hypercholesterolemia (FH) was analyzed to screen for small structural rearrangements that could not be detected by Southern blot hybridization. Three different small deletions were detected in exon 11 of 3 FH patients and were characterized by DNA sequence analysis. Of them two mutations are in‐frame 36‐bp (FH 2) and 9‐bp (FH 34) deletions that result in the loss of twelve amino acids (from Met510 to Ile521) and three amino acids (Thr513, Asp514 and Trp515), respectively. Both mutations are located in the third of the five YWTD motifs of the LDL receptor gene. The third mutation (FH 400) is a 2‐bp deletion that shifts the translational reading frame and results in a prematurely terminated receptor protein. The generation of a 36‐bp deletion can be explained by the formation of a hairpin‐loop structure mediated by inverted repeat sequences. On the other hand, the mechanism responsible for the 9‐ and the 2‐bp deletions is probably strand‐slippage mispairing mediated by short direct repeats. All of these three deletions are novel mutations. Each of the three deletions was detected only in a single pedigree out of 80 FH families analyzed.