Molecular basis of nonclassical steroid 21-hydroxylase deficiency detected by neonatal mass screening in Japan.

Since 1989, neonatal mass screening for congenital adrenal hyperplasia (CAH) has been performed in Japan, and the frequency of the classical form of 21-hydroxylase deficiency was found to be nearly identical to that in other countries. However, it has not yet been determined whether our mass screening program can detect the nonclassical (NC) form. From 1991 to 1994, about 4,500,000 infants underwent CAH mass screening in Japan. During this period, we identified by screening 2 siblings and 2 unrelated patients who had mild elevation of serum 17-hydroxyprogesterone levels at 5 days of age, but who revealed no symptoms of CAH. They were diagnosed as having probable NC steroid 21-hydroxylase deficiency. To clarify the molecular basis of NC CAH detectable by neonatal screening in Japan, the steroid 21-hydroxylase (CYP21) genes from these cases were analyzed. The 2 siblings (patients 1 and 2) had I172N and R356W mutations in 1 allele and in the other allele had local gene conversion, including the P30L mutation in exon 1. Patient 3, who was unrelated, had gene conversion encoding the same P30L mutation in 1 allele and in the other allele had an intron 2 mutation (668-12 A-->G), causing aberrant ribonucleic acid splicing, and the R356W mutation. Patient 4, also a compound heterozygote, had the R356W and 707del8 mutations. The estimated rate of detection of the NC form by mass screening (1:1,100,000) seemed low compare to the established detection rate for the classical form (1:18,000). As all of our 4 patients were compound heterozygotes with at least 1 allele bearing 1 or more mutations associated with classic CAH, it may be difficult to detect NC cases carrying only NC-associated alleles using our current neonatal mass screening methods.

[Results of genitoplasty for virilized genitalia of 41 females with congenital adrenal hyperplasia].

OBJECTIVE: The results of genitoplasty performed at our clinic for 41 female patients with congenital adrenal hyperplasia (CAH) were reviewed. PATIENTS: Among 57 females with CAH (56 with 21-hydroxylase deficiency and 1 with 11 beta-hydroxylase deficiency) diagnosed between 1961 and 1995, 41 underwent genitoplasty. For the rest of 16 patients, 1 received already clitorectomy, 11 (19%) needed no surgical reconstruction, 2 died before surgery, and 2 are in a waiting list for surgery. RESULTS: According to Prader's classification of virilized genitalia, 41 patients who received surgery included 8 of type I, 10 of type II, 17 of type III, and 4 of type IV. There was no patients of type V, and 2 were not classified because of previous surgery. Four patients underwent genitoplasty 7 times in total before visiting us. The age receiving initial genitoplasty at our clinic ranged from one year and 9 months to 20 years (median: 4 years and 5 months). Follow-up period averaged 17 years with the range of 2 months to 33 years. For enlarged clitoris, clitorectomy (amputation of clitoris) was carried out for 22 patients before 1975 and corporal resection for 17 after 1975. Two patients manifested recurrence of clitoromegaly after the surgery because of either incomplete resection of the clitoris or patient's ignorance of taking glucocorticoid regularly. Thirty-six patients needed labiovaginoplasty. Simple midline cutback was performed for 6 patients and Y-V plasty (skin flap labiointroitoplasty) for 30. Vaginal orifice was not identified in 5 patients at the initial surgery. Two patients received successful second introitoplasty at the older age than 10. Post-operative stenosis of vaginal orifice was confirmed in 6 patients and 3 underwent second introitoplasty. Among 23 patients older than 25 years at present, 7 got married and 4 raised 6 children with assistance of Caesarian section. CONCLUSIONS: Most of female patients with CAH needed genital plastic surgery including clitoroplasty and labiovaginoplasty. Corporal resection for enlarged clitoris with sufficient hormone therapy brought a satisfactory result. Y-V plasty for labiovaginoplasty proved to be an excellent method, especially from cosmetic viewpoint, but remained some problems as one stage operation including difficult identification of vaginal orifice and post-operative stenosis of introitus in some patients.

[Disturbance of active sleep components in neonates with congenital hypothyroidism].

In order to investigate the effect of hypothyroidism during the early development on the functional brainstem maturation, polysomnograms were recorded on 7 patients with congenital hypothyroidism detected by neonatal screening before treatment. The following two sleep indices for phasic muscle activity during rapid eye movement (REM) sleep were evaluated: dissociation index (DI) and % body movements in REMs burst (% BM). The DI is defined as the ratio of the number of twitch movements (TMs) during REM sleep to the sum of TMs and localized movements (LMs) during REM sleep. The % BM is the percentage of TMs and LMs which occur during bursts of REMs in relation to the sum of TMs and LMs during REM sleep. The DI and % BM can reflect maturation of the tonic and phasic inhibitory system functioning during REM sleep, respectively. In congenital hypothyroidism, DI was lower than that in controls, while % BM was identical. The tonic inhibitory system was specifically involved, whereas the phasic one was preserved. It is suggested that thyroid hormone could play an important role on the functional brainstem maturation. We propose to investigate their neuropsychological development over the long term to elucidate the influence of hypothyroidism.

Urinary pregnanetriol-3-glucuronide in children: age-related change and application to the management of 21-hydroxylase deficiency.

Urinary concentrations of pregnanetriol-3-glucuronide (PT-3-G) were determined in 485 normal Japanese subjects (277 males and 208 females), aged 5 days to 20 years, using an enzyme-linked immunosorbent assay (ELISA). The usefulness of urinary PT-3-G concentrations before giving the morning dose of medications in monitoring the adequacy of glucocorticoid treatment was assessed in eight patients with 21-hydroxylase deficiency (21-OHD). The ratio of PT-3-G to excreted creatinine (PT-3-G/Cre ratio) increased significantly during the first month and did not change from age 1 month to 1 year of life. The ratio decreased to a nadir at age 3 or 4 years followed by continuous, significant increase until late adolescence. In the subjects treated with corticosteroids for 21-OHD, PT-3-G/Cre ratios at the 50th percentile or below suggested a risk of excessive treatment, as judged by the patients' growth. Measurement of the PT-3-G/Cre ratio enabled recognition of corticosteroid overtreatment, which was not demonstrated by determining the serum concentrations of 17 alpha-hydroxyprogesterone (17-OHP). On the other hand, ratios at the upper 95-99% tolerance limits seemed to be required for optimal control. The present study revealed the normal age-related changes in urinary excretion of PT-3-G and showed it to be a reliable marker for evaluating glucocorticoid treatment in young children with 21-OHD.

[A case of neonatal meningitis due to Flavobacterium meningosepticum successfully treated with cefmetazole and cefotaxime].

A 3-day-old male infant, weighing 3,413 g and a gestational age of 38 weeks developed neonatal meningitis due to Flavobacterium meningosepticum. Treatment with cefmetazole and cefotaxime led him to a complete recovery without neurologic deficit. Of 82 previously published cases under 1 year old, 41 cases died and 16 of survivors developed hydrocephalus because the organism was resistant to many antibiotics. Therapy of meningitis due to the organism has not been standardized but the early laboratory identification and the choice of effective and safe antibiotics determined by antimicrobial sensitivity test improve the outcome.

[Fundamental and clinical studies of cefaclor in oral surgery].

Fundamental and clinical studies on cefaclor (CCL) have been performed and the following results were obtained. CCL was orally administered to NZW rabbits at the dose of 20 mg/kg, and its concentrations in blood and various tissues of oral organs were determined. Pattern of change in its blood concentration after the administration was similar to that of change in its concentration in the tissues of oral organs. Its concentration in blood was the highest followed by gingiva, parotid gland, submandibular gland, cervical lymph node and tongue in descending order. Comparative studies of CCL against cephalexin (CEX) were conducted in 5 healthy volunteers with cross over method. The 5 volunteers were orally given 500 mg of CCL or CEX at 1 dose after meal. Peak blood levels of CCL and CEX were 14.8 micrograms/ml at 2 hours and 11.5 micrograms/ml at 3 hours, respectively. The dose of 750 or 1,500 mg/day of CCL in 3 divided doses was orally administered to 71 patients with acute purulent infections in oral tissues for 3 to 13 days. Evaluation of effect was determined by the criteria for evaluation of antimicrobial agents in oral surgery. Out of the 70 patients, excellent clinical response was observed in 18 patients, good in 40, and poor in 12. Effective rate was 83%. In vitro antibacterial activities (MIC) of CCL and CEX were studied in 74 out of 81 strains (41 from aerobes and 40 from anaerobes) isolated from 47 patients. CCL showed stronger antibacterial activities than CEX. MICs of CCL against 30 strains of Gram-positive anaerobes were distributed from 0.10 to 3.13 micrograms/ml with a peak of 0.78 micrograms/ml. As adverse reaction due to CCL, eruption was observed in only 1 patient. Laboratory tests in 61 patients who received CCL showed elevation of GOT in 1 patient and elevation of GOT and GPT in 1 patient. From the above fundamental and clinical results, CCL was considered to be a useful antibiotic for the treatment of acute purulent infections caused by aerobes and anaerobes in oral surgery field.

[Clinical experience with cefmetazole in the field of oral surgery].

Tissue concentrations of cefmetazole (CMZ) in rabbits were 17--49% of the blood levels at the corresponding time. Transfer pattern of CMZ was very similar to the pattern of the blood level, suggesting to be supposable the tissue concentration of CMZ. The clinical trial was studied concentrating on the observation of side effects and laboratory findings, especially the change of transaminase after anesthesia with GOF (gas-oxygen-fluothane). Four cases, who had anethesia with GOF, revealed the elevation of transaminase.