Analysis of clinical phenotype and genotype of Chinese children with disorders of sex development / 中华儿科杂志

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.

Relationship between body mass index and sexual development in Chinese children / 中华儿科杂志

Objective: To investigate the relationship between body mass index (BMI) and sexual development in Chinese children. Methods: A nationwide multicenter and population-based large cross-sectional study was conducted in 13 provinces, autonomous regions and municipalities of China from January 2017 to December 2018. Data on sex, age, height, weight were collected, BMI was calculated and sexual characteristics were analyzed. The subjects were divided into four groups based on age, including ages 3-<6 years, 6-<10 years, 10-<15 years and 15-<18 years. Multiple Logistic regression models were used for evaluating the associations of BMI with sexual development in children. Dichotomous Logistic regression was used to compare the differences in the distribution of early and non-early puberty among normal weight, overweight and obese groups. Curves were drawn to analyze the relationship between the percentage of early puberty and BMI distribution in girls and boys at different Tanner stages. Results: A total of 208 179 healthy children (96 471 girls and 111 708 boys) were enrolled in this study. The OR values of B2, B3 and B4+ in overweight girls were 1.72 (95%CI: 1.56-1.89), 3.19 (95%CI: 2.86-3.57), 7.14 (95%CI: 6.33-8.05) and in obese girls were 2.05 (95%CI: 1.88-2.24), 4.98 (95%CI: 4.49-5.53), 11.21 (95%CI: 9.98-12.59), respectively; while the OR values of G2, G3, G4+ in overweight boys were 1.27 (95%CI: 1.17-1.38), 1.52 (95%CI: 1.36-1.70), 1.88 (95%CI: 1.66-2.14) and in obese boys were 1.27 (95%CI: 1.17-1.37), 1.59 (95%CI: 1.43-1.78), and 1.93 (95%CI: 1.70-2.18) (compared with normal weight Tanner 1 group,all P<0.01). Analysis in different age groups found that OR values of obese girls at B2 stage and boys at G2 stage were 2.02 (95%CI: 1.06-3.86) and 2.32 (95%CI:1.05-5.12) in preschool children aged 3-<6 years, respectively (both P<0.05). And in the age group of 6-10 years, overweight girls had a 5.45-fold risk and obese girls had a 12.54-fold risk of B3 stage compared to girls with normal BMI. Compared with normal weight children, the risk of early puberty was 2.67 times higher in overweight girls, 3.63 times higher in obese girls, and 1.22 times higher in overweight boys, 1.35 times higher in obese boys (all P<0.01). Among the children at each Tanner stages, the percentage of early puberty increased with the increase of BMI, from 5.7% (80/1 397), 16.1% (48/299), 13.8% (27/195) to 25.7% (198/769), 65.1% (209/321), 65.4% (157/240) in girls aged 8-<9, 10-<11 and 11-<12 years, and 6.6% (34/513), 18.7% (51/273), 21.6% (57/264) to 13.3% (96/722), 46.4% (140/302), 47.5% (105/221) in boys aged 9-<10, 12-<13 and 13-<14 years, respectively. Conclusions: BMI is positively correlated with sexual development in both Chinese boys and girls, and the correlation is stronger in girls. Obesity is a risk factor for precocious puberty in preschool children aged 3-<6 years, and 6-<10 years of age is a high risk period for early development in obese girls.

Chinese Siblings with Prader-Willi Syndrome Inherited from Their Paternal Grandmother

Background: Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder causedby failure of expression of paternally inherited genes in the PWS region of chromosome 15.Case characteristics: Two siblings who both met the inclusion criteria for clinical diagnosisof PWS during neonatal period. Outcome: Molecular genetic analysis demonstrated a 417-kb microdeletion within the 15q11.2 region inherited from siblings’ paternal grandmother,involving key genes of PWS, except for UBE3A, which may explain why their father andpaternal grandmother had a normal phenotype. Conclusion: The findings may be helpfulfor better understanding of the underlying mechanism of this rare imprinting defect

Effects of obesity on peak level of luteinizing hormone in gonadotropin-releasing hormone agonist test and obesity-related hormones in girls with central precocious puberty / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the effects of obesity on the peak level of luteinizing hormone (LH) in the gonadotropin-releasing hormone (GnRH) agonist test and obesity-related hormones in girls with central precocious puberty (CPP).</p><p><b>METHODS</b>Three hundred and thirty-three girls with CPP who underwent the GnRH agonist test between 2012 and 2014 were classified into three groups: normal weight (n=123), overweight (n=108), and obesity (n=102), according to body mass index (BMI). The sexual development indices were compared between the three groups. Twenty girls were randomly selected from each group for evaluation of the serum levels of leptin, sex hormone binding globulin (SHBG), neurokinin B, and kisspeptin. The correlation of BMI with the levels of various hormones was assessed using Pearson correlation analysis.</p><p><b>RESULTS</b>There was no significant difference in mean age at diagnosis between the three groups; however, the bone age was significantly higher in the overweight and obesity groups than in the normal weight group (P<0.05). The peak level of LH in the GnRH agonist test and SHBG level in the normal weight group were significantly higher than those in the overweight and the obesity groups, while the serum levels of leptin and neurokinin B were significantly lower in the normal weight group than in the overweight and the obesity groups (P<0.05). BMI was negatively correlated with the peak level of LH in the GnRH agonist test and SHBG level (P<0.05), and positively correlated with the levels of leptin and neurokinin B (P<0.05).</p><p><b>CONCLUSIONS</b>The effects of BMI on the result of the GnRH agonist test and levels of obesity-related hormones should be taken into account in girls with precocious puberty.</p>

A clinical follow-up study of premature thelarche in infants under two years of age / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the clinical status and natural course of premature thelarche (PT) in infants under 2 years of age and to analyze the predictive factors for regression of thelarche.</p><p><b>METHODS</b>The clinical and laboratory data of 863 infants under 2 years of age, who visited the department of endocrinology in our hospital due to PT between October 2009 and September 2010, were analyzed. A a longitudinal follow-up study was performed.</p><p><b>RESULTS</b>Of the infants under 2 years of age with isolated PT, 89.3% showed a regression before the age of 3 years (mean 17±5.6 months), 10.7% had recurrent or persistent thelarche, with no regression after the age of 3 years, and some even developed into central precocious puberty. The independent predictive factors for regression of thelarche were Tanner stage at the first visit and whether baseline estradiol level had increased.</p><p><b>CONCLUSIONS</b>PT in infants under 2 years of age is not rare in the clinical setting, and it usually runs a self-limited course, subsiding before the age of 3 years. However, regular follow-ups should be performed for infants aged over 2 years with persistent thelarche.</p>

Detection of thyroid antibodies in children with type 1 diabetes mellitus / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of positive thyroid antibodies in children with type 1 diabetes mellitus (T1DM) and its influencing factors.</p><p><b>METHODS</b>The clinical data of T1DM children who were treated in the Children's Hospital of Zhejiang University from May 2005 to April 2011 were retrospectively studied. The relationships of thyroid globulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb) with cytokines IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ were evaluated, and the percentages of CD3+, CD4+ and CD8+ T-lymphocytes in peripheral blood were examined.</p><p><b>RESULTS</b>A total of 186 T1DM children with complete data of both TGAb and TPOAb were included in the study, among whom 143 with normal TGAb and TPOAb levels and 43 (23.1%) presented with positive thyroid antibody (including 21 cases with both positive TGAb and positive TPOAb). Eighteen cases (9.7%) were diagnosed as autoimmune polyglandular syndrome type 3 variant (APS3v). Significantly more patients in the positive thyroid antibody group had a family history of diabetes than in the negative thyroid antibody group (27.9% vs 14.7%; P<0.05). The average age of the positive thyroid antibody group was 10.1±3.2 years, which was significantly greater than that in the negative thyroid antibody group (8.1±4.0 years) (P<0.05). The IL-2 level (4.48 ±1.27 pg/mL vs 2.82 ±0.84 pg/mL, P<0.05) and the percentage of peripheral CD3+ T-lymphocyte[(61±11)% vs (66±11)%; P<0.05] were also different between the positive and negative thyroid antibody groups.</p><p><b>CONCLUSIONS</b>Genetic background and abnormal function of T-lymphocytes (especially higher IL-2 level) may be involved in the elevated prevalence of positive thyroid antibody in T1DM children.</p>

A 10-year review of childhood type 1 diabetes mellitus and the clinical value of interleukin-10 in diabetic ketoacidosis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To review the incident status of childhood type 1 diabetes mellitus hospitalized in the Children's Hospital of Zhejiang University School of Medicine from 1999 to 2009 and to explore the clinical value of IL-10 in diabetic ketoacidosis.</p><p><b>METHODS</b>The clinical data of 263 children with type 1 diabetes mellitus hospitalized in the Children's Hospital of Zhejiang University School of Medicine from January 1999 to February 2009 were retrospectively reviewed. Serum lipid levels were measured in 48 children with type 1 diabetes mellitus and in 24 healthy children. The diabetic children were classified into two subgroups, with or without ketoacidosis. Serum lipid and cytokines levels were compared.</p><p><b>RESULTS</b>Childhood type 1 diabetes mellitus was common in females (56.3%). The peak incident age of the disease was between 6 and 11.9 years. Diabetic ketoacidosis was as the presenting symptom for the first visit in 86 cases (32.7%). The levels of serum lipid, blood glucose and HbA1c in diabetic children with ketoacidosis were significantly higher than those without ketoacidosis (P<0.05). Logistic analysis demonstrated that the increased levels of blood glucose, serum lipid and HbA1c were risk factors for diabetic ketoacidosis. The level of serum IL-10 in diabetic children with ketoacidosis was significantly higher than that in patients without ketoacidosis (P<0.01), while there were no differences in serum levels IL-2, IL4, IL-6, TNF-α and IFN-γ between them. Serum levels IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ in diabetic children were significantly higher than those in healthy children (P<0.01).</p><p><b>CONCLUSIONS</b>Ketoacidosis is a common acute complication of type 1 diabetes mellitus. The disorders of glucose and lipid metabolism are the risk factors for ketoacidosis in diabetic children. IL-10 may be a sensitive index of diabetic ketoacidosis in children with type 1 diabetes mellitus.</p>

Efficacy and safety of recombinant human growth hormone solution in children with growth hormone deficiency in China: a multicenter trial / 中华儿科杂志

<p><b>OBJECTIVE</b>Human growth hormone (hGH) is an essential therapeutic drug for the treatment of growth hormone (GH) deficiency (GHD). However, the process of dissolving hGH of the powder form is complicated and potentially hazardous. In the present study, we evaluated the efficacy and safety of preparation in the replacement therapy for children with GH deficiency.</p><p><b>METHODS</b>A 12-month randomized, open-label, multicenter trial was conducted in 31 previously untreated children with growth failure secondary to GH deficiency [20 boys and 11 girls, mean age (10.5 +/- 4.1) years]. An recombined human growth hormone (rhGH) solution (Iintropin AQ) was given via subcutaneous injection daily in every evening at a weekly dose of 0.25 mg/kg. The patients were followed up at 3, 6, 9, and 12 months of the treatment, and the course of treatment was 12 months. Body height was measured 3-monthly and height velocity (HV) and mean height standard deviation score (HT SDS) were calculated. Serum Insulin-like growth factor I (IGF-1), Insulin-like growth factor binding protein 3 (IGFBP-3), GH antibodies and safety parameters were assessed at the baseline and at 3-month intervals. Bone age (BA) was assessed at the baseline and the rate of skeletal maturation (DeltaBA/DeltaCA) was calculated after 6 and 12 months of rhGH treatment by a central bone age reader. Moreover, the safety of rhGH solution treatment was assessed.</p><p><b>RESULTS</b>After 12 months of liquid rhGH therapy, growth parameters were significantly increased over baseline. (1) The mean (+/- SD) height increment DeltaHT (cm) was 4.0 +/- 1.3, 7.0 +/- 2.0, 10.3 +/- 2.6 and 12.9 +/- 3.3 after 3, 6, 9, and 12 months of treatment, respectively (P < 0.01), which indicated linear growth after treatment. The GV (cm/years) was 2.7 +/- 0.9 before treatment and increased to 16.0 +/- 5.1, 14.1 +/- 4.0, 13.7 +/- 3.5, and 12.9 +/- 3.3 after treatment, suggesting that catch-up growth was significant after treatment as compared to the pre-treatment status (P < 0.01). Accordingly, post-treatment catch-up growth was obvious, significant differences were observed in HT SDS, which was -4.62 +/- 1.46 at the onset of therapy and increased significantly after the treatment to -3.80 +/- 1.53, -3.28 +/- 1.60, -2.86 +/- 1.75 and -2.47 +/- 1.86, respectively (P < 0.01). The height difference between GH deficient children and unimpaired children of the same age and gender gradually decreased after treatment, which was significantly different from that seen before treatment (P < 0.01). (2) The levels of serum IGF-1 and IGFBP-3 were increased comparably for the treatment. IGF-1 level (microg/L) was 41 +/- 64 at baseline and increased to 179 +/- 155, 202 +/- 141, 156 +/- 155 and 159 +/- 167 after 3, 6, 9, 12 months of treatment. IGFBP-3 level (mg/L) was 1540 +/- 1325 at baseline, and increased to 3891 +/- 1815, 4051 +/- 1308, 3408 +/- 1435 and 3533 +/- 1413, respectively, suggesting that with the increases in height, IGF-1, and IGFBP-3 were significantly activated to relatively high levels by the medication and reached peak values between 3 and 6 months of treatment. The levels of IGF-1 and IGFBP-3 were significantly different before and after treatment (P < 0.01). The IGF-1/IGFBP-3 molar ratio significantly increased during GH therapy (0.143 +/- 0.013 pre-therapy up to 0.240 +/- 0.055 post-therapy, P < 0.01). The IGF-1/IGFBP-3 molar ratio tended to stabilize after 3-month GH therapy. (3) The bone age assessment carried out 6 and 12 months after treatment showed that the bone maturity (DeltaBA/DeltaCA) was 1.01 +/- 0.57 and 1.07 +/- 0.75, respectively, suggesting that there was no speed-up development in the bone age. No severe adverse events were observed during the trial and the most frequent accompanying event was mild hypothyroidism.</p><p><b>CONCLUSIONS</b>rhGH solution (Iintropin AQ) is a safe and effective preparation in the replacement therapy for children with GH deficiency.</p>

Nonalcoholic steatohepatitis in obese children: the prevalence and possible mechanism / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the prevalence of nonalcoholic steatohepatitis (NASH) in obese children and its possible mechanism.</p><p><b>METHODS</b>Three subgroups were classified according to their body mass index (BMI) in 123 obese children with BMI over 23 aged 7 to 16:34 cases with BMI> or =31 group; 57 cases with 25< or =BMI<30 group; 32 cases with 23< or =BMI<25 group. Ultrasonographic and biochemical parameters including serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum cholesterol, serum triglyceride, serum uric acid and free glucose to free insulin ratio (FGIR) were assayed. Twenty four children suspected as benign acanthosis nigricans underwent skin biopsy and its association with nonalcoholic steatohepatitis was also discussed.</p><p><b>RESULTS</b>Ninety-nine children (80.49 %) showed abnormal hepatic sonograms and 54 were diagnosed as NASH with the prevalence of 43.90%. Compared with the other two groups, BMI> or =31 group was significantly higher in prevalence of abnormal hepatic sonograms, NASH, decreased FGIR and risk of benign acanthosis nigricans (P<0.01). Fifty-four children diagnosed as NASH showed significantly higher incidence of hyperlipidemia, insulin resistance and higher body mass index as compared with 24 subjects without fatty liver changes. In 54 NASH children, 20(37.04%) had benign acanthosis nigricans. By bivariate analysis, ALT and AST were correlated well with BMI, cholesterol, triglyceride and FGIR (r(s)=0.413, 0.290, 0.379, -0.477, P<0.01; r(s)=0.359, 0.349, 0.348, -0.369, P<0.01).</p><p><b>CONCLUSION</b>There is a high prevalence of nonalcoholic steatohepatitis in simple obese children and high incidence of benign acanthosis nigricans in NASH subjects. BMI> or=30 is a high risk factor of being NASH. Severe disturbance of lipid metabolism and insulin resistance may be involved in the mechanism of NASH.</p>

Effect of recombinant human growth hormone on glucose metabolism in children with growth hormone deficiency / 中华儿科杂志

<p><b>OBJECTIVE</b>Numerous studies in children with growth hormone deficiency (GHD) show that recombinant human growth hormone (rhGH) treatment results in significant catch-up growth, but some papers reported that the children who underwent rhGH therapy might be at increased risk of diabetes. The aim of this study was to investigate the effects of rhGH treatment on blood glucose and insulin metabolism in children with GHD and the relationship between growth hormone (GH) and glucose homeostasis.</p><p><b>METHODS</b>In this study, 44 children with GHD treated with rhGH [0.1 U/(kgxd)] and age- and sex-matched 20 healthy children were enrolled. The GHD group included 28 males and 16 females aged from 4.5 to 16.5 years (mean 10.4 +/- 2.6 years), including 18 cases of complete GHD and 26 cases of partial GHD. The sexual development stage of all subjects was in Tanner I. Oral glucose tolerance tests (OGTT) were done, and body mass index (BMI), serum insulin-like growth factor-1 (IGF-1) level and insulin resistance by homeostasis model (HOMA-IR) were measured at the time of diagnosis and every 3 months after rhGH therapy. Continuous glucose monitoring system (CGMS) was applied for two cases with hyperglycemia.</p><p><b>RESULTS</b>(1) Fasting glucose and IGF-1 levels increased since 3 months of treatment and did not decrease since then. The levels of fasting glucose and IGF-1 at every time points of rhGH therapy were higher than the levels at the time of diagnosis (F = 6.81, P < 0.01; F = 7.31, P < 0.01, respectively). HOMA-IR and fasting insulin levels were increased since 3 and 9 months of treatment (P = 0.001 and P = 0.021, respectively). They decreased after 12 months of therapy and the levels at 18 months of therapy were similar to that at diagnosis. (2) Pearson correlation analysis showed that HOMA-IR was positively correlated with BMI, IGF-1 and the duration of treatment (r = 0.251, 0.437, 0.281, P < 0.01, respectively). The curve between HOMA-IR and duration of therapy was similar with parabola and the quadratic equation obtained was as follows: HOMA-IR = 1.5048 + 0.2177 x duration of therapy (months)-0.0103 x duration of therapy (months)(2) (r(2) = 0.147, F = 14.16, P < 0.01). (3) Two cases had transitory hyperglycemia. Their fasting glucose levels were all higher than 7.1 mmol/L. The glucose levels returned to normal after 1 month and 5 days respectively. OGTT and CGMS showed that their plasma glucose levels were normal after rhGH therapy was applied again.</p><p><b>CONCLUSION</b>The children who underwent rhGH therapy may be at increased risk of insulin resistance (especially during the first year) and the therapy may even induce transitory glucose metabolic disorder in a very small proportion of patients. Circulating IGF-1 may participate in the control of insulin sensitivity and play an important role in the hormonal balance between GH and insulin. It may be necessary to monitor glucose metabolism and IGF-1 for all children who are treated with rhGH therapy.</p>

Determination of six major human herpes viruses in cerebrospinal fluid and blood of children with consensus primers / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To identify 6 major human herpesviruses with consensus primers and to explore its clinical application.</p><p><b>METHODS</b>Based on the highly-homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1, type 2, Epstein-Barr virus and cytomegalovirus; and another was used to amplify varicella-zoster virus or human herpesvirus 6. Virus species identification was performed by restriction enzyme digestion with BamH I and BstU I. Thirty-eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for herpes virus DNA using the PCR-RFLP assay with these primers.</p><p><b>RESULTS</b>Thirteen out of 38 CSF specimens (34.2%) were herpes virus positive. All blood specimens from 27 confirmed cases showed positive results, while for 22 clinically diagnosed cases 16 (72.7%) were positive. The types of herpes virus were determined using restriction enzyme digestion with BamH I and BstU I. Two CSF specimens from the patients, who were treated with aciclovir for 2 - 3 days, were still positive for herpes virus DNA by this method. None of the control blood or CSF controls were positive for herpesvirus by PCR.</p><p><b>CONCLUSION</b>The PCR-RFLP method used in this study is a specific, sensitive and practicable one for diagnosis of herpes virus infection.</p>

Obese children with benign acanthosis nigricans and insulin resistance: analysis of 19 cases / 中华儿科杂志

<p><b>OBJECTIVE</b>The prevalence of obesity and of type 2 diabetes mellitus in children have increased in the Chinese population over the past two decades, and thus diabetes prevention has become a major concern of public health agencies. Identification of individuals at risk for diabetes is an essential first step in designing and implementing intervention programs. Insulin resistance is the hallmark of the pathophysiology of type 2 diabetes mellitus. Subjects with hyperinsulinemia and impaired glucose tolerance are well accepted as being at high risk for diabetes. Acanthosis nigricans (AN) has been proposed as a reliable marker of hyperinsulinemia, but its utility for predicting hyperinsulinemia has not been systematically evaluated in obese children. In order to further explore the relationship between obese childhood with benign acanthosis nigricans and insulin resistance and type 2 diabetes mellitus, we examined 19 obese children with benign acanthosis nigricans.</p><p><b>METHODS</b>Nineteen of seventy six obese children (25%) with BMI over 25 enrolled in the Children' Hospital of Zhejiang University School of Medicine fromJune 1st to September 1st in 2003 were studied. Skin biopsies were performed in these 19 obese children with acanthosis nigricans for final diagnosis. Levels of glucose, insulin, and glucose/insulin ratio were measured on fasting blood specimens and anthropometric parameters including waist/hip ratio, fat mass, body fat percentage and body mass index were examined. Oral glucose tolerance tests were also performed in these 19 children with benign acanthosis nigricans.</p><p><b>RESULTS</b>Anthropometric parameters including waist/hip ratio, fat mass, body fat percentage and body mass index as well as fasting insulin level in acanthosis nigricans group were significantly higher than that of healthy controls (P < 0.01). Fasting glucose to insulin ratio (FGIR) of these 19 obese children with benign acanthosis nigricans was 4.27 +/- 0.53, indicating apparent insulin resistance. One of them was diagnosed as type 2 diabetes mellitus and ten of them showed impaired oral glucose tolerance.</p><p><b>CONCLUSION</b>Childhood benign acanthosis nigricans is tightly associated with obesity, hyperinsulinemia, insuline resistance and type 2 diabetes mellitus, and may be used as a reliable index of insulin resistance.</p>

Detection of four human herpesviruses DNA and virus-specific IgM antibody in blood specimens of infants / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish a restriction endonuclease pattern which could detect and differentiate four major human herpesviruses by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequence analysis.</p><p><b>METHODS</b>A pair of primer, which was designed according to sequences in well-conserved regions of the DNA polymerase gene in human herpesviruses, was designed to amplify herpes simplex virus type 1 and 2 (HSVI/II), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Sequences of the primers are as follows: P(1) (5'-CGACTTTGCCAGCCTGACC-3') and P(2) (5'-AGTCCGTGTCCCCGTAGATG-3'). DNA of four strains of standard herpesviruses were amplified by PCR, and further studied by DNA cloning, sequence analysis and RFLP. At last, the authors established the PCR-RFLP technique to differentiate the four different herpesviruses. Meanwhile, 75 clinical blood specimens from infants with suspected viral infection and 38 blood specimens from healthy children were evaluated for herpesviruses DNA or virus-specific IgM antibody by PCR-RFLP or by ELISA.</p><p><b>RESULTS</b>The PCR amplified products of four human herpesviruses were from 510 bp to 592 bp in length and were analyzed for herpesvirus types with restriction endonuclease technique. The specificity and sensitivity of this PCR-RFLP were examined. There was no cross-reaction with Escherichia coli, Staphylococcus aureus, hepatitis B virus (HBV), Clostridium neoformans and human-genomic DNA and the lowest detection level was 0.1 fg DNA. Among 75 specimens, 23 were positive by PCR and the positive rate was 30.7%, including 13 for CMV, four for EBV, five for HSVII and one for HSVI after restriction enzyme digestion with BamHI and BstUI, while 10 were positive by ELISA and positive rate was 13.3%. All ELISA-positive specimens were likewise positive by PCR. Thirteen of 65 specimens that were ELISA-negative were tested positive by PCR. An infant with CMV infection was determined with viral DNA and virus-specific IgM antibody in blood at 3, 4 and 6 months after birth, respectively. The result showed that she was still CMV DNA-positive in blood whereas IgM antibody was positive only at month 3 after birth. None of the 38 control blood specimens was positive for herpesvirus by this PCR-RFLP or by ELISA.</p><p><b>CONCLUSIONS</b>This PCR-RFLP technique was specific, sensitive, rapid and accurate in diagnosing herpesviruses infection in infants, and it could detect herpesviruses DNA in specimens which were negative for IgM antibody by ELISA.</p>

Simplified gonadorelin stimulation test in diagnosis of precocious puberty / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To assess the diagnostic value of the simplified gonadorelin stimulation test for precocious puberty.</p><p><b>METHODS</b>Two hundred and ninety-two girls with signs of advanced breast development received the gonadorelin stimulation test. According to the result of gonadorelin stimulation test, the girls were divided into 3 groups: 151 with central precocious puberty (CPP),119 with premature thelarche (PT) and 22 with peripheral precocious puberty (PPP).</p><p><b>RESULTS</b>LH or FSH levels at 15 min, 30 min, 60 min in PPP group were not significantly different (P>0.05). Those were significantly different in PT group (P<0.01). The highest levels of LH were at 30 min and the highest levels of FSH were at 60 min. LH or FSH levels at 15 min, 30 min, 60 min in CPP group were significantly different (P<0.01) with the highest levels at 30 min. The ratio of basal LH and FSH >0.2 had a diagnostic sensitivity of 48.3 % and specificity of 69.7%. Taking the LH/FSH ratio >0.9 at 15 min, 30 min, 60 min as cut-off value, the diagnostic sensitivity was 80.1%, 68.9% and 38.4%, and the specificity was 90.8%, 96.6% and 69.7%, respectively.</p><p><b>CONCLUSION</b>The LH/FSH ratio>0.9 at 15 min after gonadorelin stimulation test can be used as a cut-off value to differentiate CPP from PT and blood sample at 60 min were not necessary.</p>

Molecular diagnosis of the specific DNA patterns of 16S-23S rRNA gene of bacteria / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.</p><p><b>METHODS</b>A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.</p><p><b>RESULTS</b>The 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.</p><p><b>CONCLUSIONS</b>The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.</p>