SLC1A4 mutations cause a novel disorder of intellectual disability, progressive microcephaly, spasticity and thin corpus callosum.

Two unrelated patients, presenting with significant global developmental delay, severe progressive microcephaly, seizures, spasticity and thin corpus callosum (CC) underwent trio whole-exome sequencing. No candidate variant was found in any known genes related to the phenotype. However, crossing the data of the patients illustrated that they both manifested pathogenic variants in the SLC1A4 gene which codes the ASCT1 transporter of serine and other neutral amino acids. The Ashkenazi patient is homozygous for a deleterious missense c.766G>A, p.(E256K) mutation whereas the Ashkenazi-Iraqi patient is compound heterozygous for this mutation and a nonsense c.945delTT, p.(Leu315Hisfs*42) mutation. Structural prediction demonstrates truncation of significant portion of the protein by the nonsense mutation and speculates functional disruption by the missense mutation. Both mutations are extremely rare in general population databases, however, the missense mutation was found in heterozygous mode in 1:100 Jewish Ashkenazi controls suggesting a higher carrier rate among Ashkenazi Jews. We conclude that SLC1A4 is the disease causing gene of a novel neurologic disorder manifesting with significant intellectual disability, severe postnatal microcephaly, spasticity and thin CC. The role of SLC1A4 in the serine transport from astrocytes to neurons suggests a possible pathomechanism for this disease and implies a potential therapeutic approach.

Novel WWOX deleterious variants cause early infantile epileptic encephalopathy, severe developmental delay and dysmorphism among Yemenite Jews.

The human WW Domain Containing Oxidoreductase (WWOX) gene was originally described as a tumor suppressor gene. However, recent reports have demonstrated its cardinal role in the pathogenesis of central nervous systems disorders such as epileptic encephalopathy, intellectual disability, and spinocerebellar ataxia. We report on six patients from three unrelated families of full or partial Yemenite Jewish ancestry exhibiting early infantile epileptic encephalopathy and profound developmental delay. Importantly, four patients demonstrated facial dysmorphism. Exome sequencing revealed that four of the patients were homozygous for a novel WWOX c.517-2A > G splice-site variant and two were compound heterozygous for this variant and a novel c.689A > C, p.Gln230Pro missense variant. Complementary DNA sequencing demonstrated that the WWOX c.517-2A > G splice-site variant causes skipping of exon six. A carrier rate of 1:177 was found among Yemenite Jews. We provide the first detailed description of patients harboring a splice-site variant in the WWOX gene and propose that the clinical synopsis of WWOX related epileptic encephalopathy should be broadened to include facial dysmorphism. The increased frequency of the c.517-2A > G splice-site variant among Yemenite Jews coupled with the severity of the phenotype makes it a candidate for inclusion in expanded preconception screening programs.

Mutations in AIFM1 cause an X-linked childhood cerebellar ataxia partially responsive to riboflavin.

BACKGROUND: AIFM1 encodes a mitochondrial flavoprotein with a dual role (NADH oxidoreductase and regulator of apoptosis), which uses riboflavin as a cofactor. Mutations in the X-linked AIFM1 were reported in relation to two main phenotypes: a severe infantile mitochondrial encephalomyopathy and an early-onset axonal sensorimotor neuropathy with hearing loss. In this paper we report two unrelated males harboring AIFM1 mutations (one of which is novel) who display distinct phenotypes including progressive ataxia which partially improved with riboflavin treatment. METHODS: For both patients trio whole exome sequencing was performed. Validation and segregation were performed with Sanger sequencing. Following the diagnosis, patients were treated with up to 200 mg riboflavin/day for 12 months. Ataxia was assessed by the ICARS scale at baseline, and 6 and 12 months following treatment. RESULTS: Patient 1 presented at the age of 5 years with auditory neuropathy, followed by progressive ataxia, vermian atrophy and axonal neuropathy. Patient 2 presented at the age of 4.5 years with severe limb and palatal myoclonus, followed by ataxia, cerebellar atrophy, ophthalmoplegia, sensorineural hearing loss, hyporeflexia and cardiomyopathy. Two deleterious missense mutations were found in the AIFM1 gene: p. Met340Thr mutation located in the FAD dependent oxidoreductase domain and the novel p. Thr141Ile mutation located in a highly conserved DNA binding motif. Ataxia score, decreased by 39% in patient 1 and 20% in patient 2 following 12 months of treatment. CONCLUSION: AIFM1 mutations cause childhood cerebellar ataxia, which may be partially treatable in some patients with high dose riboflavin.

Synchronizing activity of basal ganglia and pathophysiology of Parkinson's disease.

Early physiological studies emphasized changes in the discharge rate of basal ganglia in the pathophysiology of Parkinson's disease (PD), whereas recent studies stressed the role of the abnormal oscillatory activity and neuronal synchronization of pallidal cells. However, human observations cast doubt on the synchronization hypothesis since increased synchronization may be an epi-phenomenon of the tremor or of independent oscillators with similar frequency. Here, we show that modern actor/ critic models of the basal ganglia predict the emergence of synchronized activity in PD and that significant non-oscillatory and oscillatory correlations are found in MPTP primates. We conclude that the normal fluctuation of basal ganglia dopamine levels combined with local cortico-striatal learning rules lead to noncorrelated activity in the pallidum. Dopamine depletion, as in PD, results in correlated pallidal activity, and reduced information capacity. We therefore suggest that future deep brain stimulation (DBS) algorithms may be improved by desynchronizing pallidal activity.

Creatine transporter deficiency: Novel mutations and functional studies.

X-linked cerebral creatine deficiency (MIM 300036) is caused by deficiency of the creatine transporter encoded by the SLC6A8 gene. Here we report three patients with this condition from Israel. These unrelated patients were evaluated for global developmental delays and language apraxia. Borderline microcephaly was noted in one of them. Diagnosis was prompted by brain magnetic resonance imaging and spectroscopy which revealed normal white matter distribution, but absence of the creatine peak in all three patients. Biochemical testing indicated normal plasma levels of creatine and guanidinoacetate, but an increased urine creatine/creatinine ratio. The diagnosis was confirmed by demonstrating absent ([14])C-creatine transport in fibroblasts. Molecular studies indicated that the first patient is hemizygous for a single nucleotide change substituting a single amino acid (c.619 C > T, p.R207W). Expression studies in HeLa cells confirmed the causative role of the R207W substitution. The second patient had a three base pair deletion in the SLC6A8 gene (c.1222_1224delTTC, p.F408del) as well as a single base change (c.1254 + 1G > A) at a splicing site in the intron-exon junction of exon 8, the latter occurring de novo. The third patient, had a three base pair deletion (c.1006_1008delAAC, p.N336del) previously reported in other patients with creatine transporter deficiency. These three patients are the first reported cases of creatine transporter deficiency in Israel.

Staining clinical specimens for acid-fast bacilli by means of a mechanical conveyor system.

A Cyto-Tek staining machine designed for Papanicolau staining of cervical smears has been adapted for auramine staining of smears of sputum and other clinical specimens for acid-fast bacilli. The results compared favourably with those obtained by a manual method of staining. Contamination of negative smears by acid-fast bacilli from positive smears was not found. Since this study the machine has continued to be in routine use with a considerable saving in labour.

An evaluation of the Technicon Autoanalyzer for automating complement-fixation tests.

Obviously there is need for a reliable means of automating complement-fixation tests. This paper offers an account of experience with an AutoAnalyzer adapted for this purpose. Results of syphilis screening tests done by the AutoAnalyzer compared with those of tests performed manually showed satisfactory agreement, the automated method being slightly less specific in our hands. There were no false negative results with the AutoAnalyzer if both Maltaner and Reiter protein antigens were used. The current design of the AutoAnalyzer contains three major shortcomings if it is to be used for large numbers of complement-fixation tests. First and foremost is the relatively slow sampling rate compatible with adequate sample discrimination. Second is the necessity for frequently replenishing the machine with samples. Third is the need for adjusting or cleaning the flow-cell during use.

Improved immunofluorescence techniques with microplates for the detection of M and G immunoglobulins against rotavirus.

A selection of microplates and an inverted microscope were used for the detection of immuno-fluorescence in large numbers of infected cell monolayers. The optical features of this instrument in combination with either Terasaki or Microlymp plates, simplify procedures and allow economical use of reagents, for determining fluorescent antibody levels. The techniques described were applied for estimating M and G immunoglobulins against rotavirus in elderly patients and the validity of the tests was established.

Diagnosis and prevalence of persistent chlamydia infection in infertile women: tissue culture, direct antigen detection, and serology.

Specimens for chlamydial culture, direct fluorescent antibody (DFA) test, two enzyme immunoassays (EIA) for antigen detection, and serum for chlamydial antibodies were collected from 256 infertile women. Specimens were taken from the tubes during tuboplasty and from the cervix and endometrium during laparoscopy or tuboplasty. Antibodies to Chlamydia trachomatis were found four times more often in patients with signs of prior pelvic inflammatory disease (PID) than in infertile women with normal pelvic findings. Only 48 (37%) of 131 patients with signs of prior PID had a history of PID. Ten or more C. trachomatis elementary bodies (EBs) per smear were found in 21 (8.2%) of 256 patients. Six patients had a positive culture or a positive antigen EIA test. All six had high numbers of EBs in the DFA test. We conclude that routine culture and EIA antigen tests detect only a minority of persistent chlamydia infections in this population, but subjective factors in the interpretation of DFA methods must be considered.

Low-dose oestradiol in the treatment of urogenital oestrogen deficiency--a pharmacokinetic and pharmacodynamic study.

Twenty-four postmenopausal women with vaginal atrophy due to oestrogen deficiency were treated with 17 beta-oestradiol administered as vaginal tablets containing 10 and 25 micrograms, respectively, in a slow-release system (Vagifem, Novo Nordisk, Denmark). All the women were treated for 2 weeks with each dose in a double-blind, cross-over study. Plasma concentrations of unconjugated oestradiol and unconjugated oestrone were measured at regular intervals for 24 h on days 1 and 14 of each treatment regimen. Cytological and clinical evaluations of the vaginal and urethral epithelium were also carried out. Initially, when the epithelium was still atrophic, dose-dependent absorption of oestradiol was demonstrated. After 14 days of treatment maturation of the vaginal epithelium was seen with both regimens and the absorption of oestradiol then declined significantly on both the 10 and the 25 micrograms dose. Oestrone levels remained unchanged and gonadotrophin levels were unaffected during treatment. Vaginal cytology showed maturation on both the 10 and the 25 micrograms dose, whereas urethral cytology showed a reduction in parabasal cells that was significant only on 25 micrograms. Clinical and subjective improvement was apparent on both doses and acceptance of treatment was good.

Low-dose 17 beta-oestradiol during maintenance therapy--a pharmacokinetic and pharmacodynamic study.

The vaginal absorption of oestradiol was studied during maintenance therapy with low-dose oestradiol. Six women with severe vaginal atrophy due to oestrogen deficiency were treated with a vaginal tablet containing 25 micrograms 17 beta-oestradiol. Initially, a daily dose was given for 2 weeks, followed by maintenance therapy with twice weekly treatment for another 10 weeks. The plasma concentrations of unconjugated oestrone and oestradiol were measured before oestrogen treatment was started at the beginning of the study. After 3 months of treatment frequent plasma sampling over a period of 24 h was performed. Gonadotrophins, vaginal and urethral cytology, clinical findings and subjective symptoms were assessed at the beginning and end of the study. Plasma concentrations of unconjugated oestradiol were at all times within the limits of postmenopausal values, but showed a slight but not statistically significant elevation after 3 months compared to pretreatment values. Plasma concentrations of unconjugated oestrone were in the low postmenopausal range throughout the study. LH levels were unaffected during the study, while FSH was somewhat lowered, but still well within the postmenopausal range. Vaginal and urethral cytology showed maturation with almost complete disappearance of parabasal cells. Clinical and subjective improvement was statistically significant during the treatment period.

Transvaginal ultrasonography for identifying endometrial pathology in postmenopausal women.

The objective of this study was to evaluate the usefulness of transvaginal ultrasonography in postmenopausal women with a clinical indication for a dilatation and curettage (D&C). Of the 167 postmenopausal women included in the study, 88% were referred for a D&C because of vaginal bleeding and 12% of the women had other clinical indications such as myomas, gynecological pain or suspected gynecological tumors. Hormone replacement therapy (HRT) was used by 37% of the women. The women were examined with transvaginal ultrasonography before the D&C. The endometrial thickness and texture were used as indicators of endometrial abnormalities. The ultrasonographical findings were related to the histological diagnosis obtained from the D&C. Histologically, 31% of the women had an atrophic endometrium and the corresponding ultrasonographically mean endometrial thickness was 4.6 mm (range 0-14 mm). Endometrial cancer was histologically found in 10% of the women and the endometrial thickness of the malignant endometrium, measured by ultrasonography, was 13.9 mm (range 6-31 mm). All the malignancies were found in the group of women with vaginal bleeding, but only one was in the group of women on HRT. Histologically, endometrial hyperplasia was found in 6.5% of the women and endometrial polyps in 8.5% after the D&Cs. In these postmenopausal women it was demonstrated that if the endometrium was < 6 mm thick, no endometrial cancer was found at histopathological investigation. By using a cut-off point of 6 mm of ultrasonographically measured endometrial thickness for identification of endometrial pathology in our study, at least 50% of the D&Cs could be spared.

Plasma oestriol following vaginal administration: morning versus evening insertion and influence of food.

The aim of this trial was to study the vaginal absorption of oestriol and to investigate whether morning rather than evening oestriol administration would produce different plasma oestriol patterns. The influence of food intake on plasma oestriol levels was also investigated. Nine post-menopausal women were given 0.5 mg oestriol (ovula supplied by Leo AB, Sweden) intravaginally every evening for 16 days. Thereafter, 1 mg oestriol was given every evening for another 5 days, except on treatment days 18 and 19 when 1 mg oestriol was given in the morning instead. Venous blood samples were collected at frequent intervals on day 19 (morning administration) and a meal was allowed 4 h later. On the day 21 (evening administration), venous blood samples were taken at frequent intervals during the night and no meal was given until the next morning. Plasma concentrations of unconjugated oestriol were measured by means of a specific radioimmunoassay (RIA). A difference was seen in the plasma oestriol patterns when the results following morning and evening administration were compared. However, no significant difference as regards the total 24-h systemic availability of oestriol was observed. A minimal increase in plasma oestriol levels was seen after a meal in the case of both morning and evening intravaginal oestriol administration, possibly as a result of enterohepatic recirculation.

Effects of vaginally-administered oestriol on post-menopausal urogenital disorders: a cytohormonal study.

Forty post-menopausal women with urogenital disorders who were inpatients in the same geriatric hospital were treated with oestriol (E3) for 6 weeks. For the first 2 weeks 0.5 mg E3 (Leo AB, Sweden) was administered intravaginally every day. Over the following 4 weeks the patients received the same quantity either once or twice weekly as a maintenance dose. Oestrogen influence on the vaginal and urethral epithelium was assessed by means of the karyopyknotic index (KPI), while the degree of maturation of the vaginal epithelium was estimated visually. Urinary bacteria were cultivated. A pronounced and progressive rise in KPI was seen in both the vaginal and the urethral epithelium following daily E3 treatment. However, neither of the two maintenance dosages was sufficient to sustain the initial maturation of the vaginal and urethral epithelium induced by E3, since the KPI returned to pretreatment values within 4 weeks. The effect of E3 administration on the vaginal epithelium was overestimated by the visual assessment method. No changes were seen in urinary bacteria. Medroxyprogesterone acetate was given before and after E3 treatment. None of the women suffered from withdrawal bleeding.

The prevalence of urogenital symptoms in postmenopausal women.

Lower urogenital tract disorders, such as vaginal athropy, urethritis, dyspareunia, recurrent urinary tract infections and urinary incontinence symptoms, are more prevalent in postmenopausal women. While these disorders are attributed to the ageing process as well as estrogen deficiency, knowledge of the relationship between estrogen status and symptomatology is scarce and hard to investigate due to the complexity of the problem. Little is known about the epidemiology of urogenital symptoms and their relationship to estrogen status and treatment. Studies of the prevalence of urogenital symptoms in postmenopausal women have been rare and results divergent. Through reviewing existing literature and relating findings to our own prevalence studies of 61-, 71- and 81-year-old women, we can conclude that many of the symptoms accounted for in our study are those known to be due to the loss of estrogen and easily dealt with by estrogen therapy. However, there is a need for more adequate information about postmenopausal symptoms and the effect of estrogens, as only a minority of postmenopausal women are currently treated.

The vaginal epithelium in the postmenopause--cytology, histology and pH as methods of assessment.

In the study of postmenopausal vaginal oestrogen deficiency, an objective assessment of the vaginal epithelium is necessary. The present study was undertaken to evaluate different methods for assessing the vaginal epithelium, during atrophic and mature conditions. The vaginal epithelium was assessed by clinical examination, pH and vaginal cytology, including morphometric analysis, before and after oestrogen treatment. Biopsies of the vaginal wall were studied using descriptive histology and immunohistochemistry methods. The antigen Ki-67, a sensitive marker of cell proliferation, was detected using the monoclonal antibody MIB1. When comparing pre- and post-treatment values from the methods, a shift towards mature values was observed, but the magnitude of the shift differed to a considerable extent. Vaginal cytology, expressed as mean maturation index (MI) shifted significantly from 94/6/0 to 0/65/35. Likewise, mean pH was significantly shifted from 6.2 to mean 4.5. The increased presence of Ki-67 positive cells could be demonstrated after oestrogen treatment, but the range of data was wide, which was also found for the thickness of the epithelium and the number of cell layers. For objective assessment of the vaginal epithelium maturation index and pH can be recommended.

Prevalence of genitourinary and other climacteric symptoms in 61-year-old women.

In a population-based cohort study, 1280 women, aged 61, were interviewed regarding their genitourinary and other postmenopausal symptoms by means of an anonymous questionnaire. The group selected was to constitute all women of 61 years of age living in Uppsala county, Sweden. The response rate was 84%. All were postmenopausal women. Seventy-three percent of the women answering admitted some degree of urinary incontinence and 33% more severe degree. Forty-nine percent reported some degree of stress incontinence, 25% a more severe degree. Thirty-one percent experienced urge incontinence, 14% severely. A minority (4%), had had more than two urinary infections during the last year. The majority (67%) had changed urinating habits, going to the toilet at night and a minority complained of increased frequency of micturation (8%). Of the participating women, 59% were still sexually active, 43% had trouble with vaginal dryness and 10% with vaginal burning. Vasomotor problems such as hot flushes (30%), daily (33%) and nightly sweating (36%) were all common troubles. Forty-seven percent of the women had asked for medical help for estrogen deficiency problems, 82% were satisfied with the help they had received. Thirty-four percent were on estrogen therapy, 16% had systemic therapy 18% low dose estrogen treatment.

Prevalence of postmenopausal symptoms in two age groups of elderly women in relation to oestrogen replacement therapy.

A cross-sectional study of the whole female population of ages 71 and 81 years in a defined part of Sweden was undertaken to investigate the prevalence of oestrogen treatment and postmenopausal symptoms. A questionnaire was mailed to 2245 women, of whom 1084 (87%) aged 71 years and 611 (62%) aged 81 years left evaluable responses. Of the responding 71- and 81-year-old women 25 and 16%, respectively were receiving oestrogen, and 4 and 2% of all women of the respective age groups were on systemic treatment. Nearly half of all the women reported urinary incontinence, which was considerate for approximately half of these women. Five and 11% of the respective age groups had experienced more than two urinary tract infections (RUTI) in the last year. RUTI had occurred both in the oestrogen-treated group and in the non-treated group. Vegetative symptoms were still encountered among these elderly women. Previous fractures were frequent, being experienced after menopause by 29 and 39% of the 71- and 81-year-old women respectively. Thirty-five and 39% of the women in respective age group had sought medical help for postmenopausal symptoms. Of the women with moderate, severe or unbearable urinary incontinence, 60 and 66% of the respective age groups had sought medical help. In only few of the totals of women on oestrogen had the treatment a complete effect. Only 2 and 1% of all women in respective age group had been offered and undergone surgery for their urinary incontinence.

Oestradiol-releasing vaginal ring for treatment of postmenopausal urogenital atrophy.

A silicone vaginal ring releasing 5-10 micrograms oestradiol/24 h for a minimum of 90 days has been developed for treatment of urogenital mucosal atrophy. The efficacy, safety and acceptability of the oestradiol-releasing ring were studied in 222 postmenopausal women with symptoms and signs of atrophic vaginal mucosa. The maturation of the vaginal epithelium, as measured by cytological parameters, was significantly improved during treatment. No proliferation of the endometrium was encountered. The therapy had a significant effect on symptoms (vaginal dryness, pruritus vulvae, dyspareunia, urinary urgency) and on signs of atrophic vaginitis, with cure/improvement registered in > or = 90%. The patient acceptability was high, since > or = 90% did not report any discomfort with the ring. Almost all of the sexually active women had the ring in place during coitus and in < or = 2% of cases discomfort was noticed by them or the partner. It is concluded that a vaginal silicone ring giving a continuous release of an ultra-low dose of oestradiol is an effective and safe treatment for urogenital oestrogen deficiency. No addition of progestagen is needed.

New kinetic data on estradiol in light of the vaginal ring concept.

The principal estrogen produced by the functioning premenopausal ovary is 17 beta-estradiol. At the point of irreversible ovarian failure, at menopause, the production of estradiol decreases dramatically, which results in circulating serum levels less than 120 pmol/l. It is important to recognise the pharmacokinetic and metabolic outcomes associated with dosage and route of delivery of estrogen. One of the most promising methods of administering estrogen replacement therapy (ERT) for local effects is the estradiol vaginal ring designed for a controlled continuous low release (7.5 micrograms estradiol/24 h) over a period of 90 days. The present study was undertaken to characterise the basal endogenous turnover of estradiol in postmenopausal women. Information on the disposition of estradiol after an intravenous dose formed the base of the kinetic model. The rate of extent of absorption of estradiol was assessed after ring application. Individual serum concentrations of estradiol were analysed without subtraction of the basal estradiol levels. The results indicate a rapidly eliminated compound (plasma clearance 2 l/min) with a distribution of approximately 50 l, resulting in an efficient half-life of about 20 min. The endogenous production was highly variable (< 1-44 micrograms/24 h). The steady-state estradiol levels following ring application did not increase and were well within the normal basal estradiol range seen in untreated women. In light of the present findings, the low daily dose, the low availability of estradiol across the vaginal wall and the controlled local delivery, favour the use of the estradiol vaginal ring.