From neurons to the neuro-glio-vascular unit: Seizures and brain homeostasis in networks.

While seizures are undoubtedly neuronal events, an ensemble of auxiliary brain cells profoundly shapes synaptic transmission in health and disease conditions. Endothelial-astrocyte-pericyte assemblies at the blood-brain barrier (BBB) and neuroglia within the neuro-glio-vascular unit (NGVU) finely tune brain parenchymal homeostasis, safeguarding the ionic and molecular compositions of the interstitial fluid. BBB permeability with neuroinflammation and the resulting loss of brain homeostatic control are unifying mechanisms sustaining aberrant neuronal discharges, with temporal specificities linked to acute (head trauma, stroke, infections) and pre-existent (genetic) or chronic ( dysplasia, tumors, neurodegenerative disorders) pathological conditions. Within this research template, one hypothesis is that the topography of BBB damage and neuroinflammation could associate with symptoms, e.g., limbic structures for seizures or pre-frontal for psychiatric episodes. Another uncharted matter is whether seizure activity, without tissue lesions or sclerosis, is sufficient to promote stable cellular-level maladaptations in networks. Contingent to localization and duration, BBB damage and inflammation forecast pathological trajectories, and the concept of an epileptic NGVU could enable time-sensitive biomarkers to predict disease progression. The coherence between electrographic, imaging and molecular NGVU biomarkers could be established from the epileptogenic to the propagating zones. This paradigm shift could lead to new diagnostic and therapeutic modalities germane to specific epilepsies or when seizure activity represents a comorbidity.

Principles of proportional recovery after stroke generalize to neglect and aphasia.

BACKGROUND AND PURPOSE: Motor recovery after stroke can be characterized into two different patterns. A majority of patients recover about 70% of initial impairment, whereas some patients with severe initial deficits show little or no improvement. Here, we investigated whether recovery from visuospatial neglect and aphasia is also separated into two different groups and whether similar proportions of recovery can be expected for the two cognitive functions. METHODS: We assessed 35 patients with neglect and 14 patients with aphasia at 3 weeks and 3 months after stroke using standardized tests. Recovery patterns were classified with hierarchical clustering and the proportion of recovery was estimated from initial impairment using a linear regression analysis. RESULTS: Patients were reliably clustered into two different groups. For patients in the first cluster (n = 40), recovery followed a linear model where improvement was proportional to initial impairment and achieved 71% of maximal possible recovery for both cognitive deficits. Patients in the second cluster (n = 9) exhibited poor recovery (<25% of initial impairment). CONCLUSIONS: Our findings indicate that improvement from neglect or aphasia after stroke shows the same dichotomy and proportionality as observed in motor recovery. This is suggestive of common underlying principles of plasticity, which apply to motor and cognitive functions.

Refractory and super-refractory status epilepticus in adults: a 9-year cohort study.

OBJECTIVE: While status epilepticus (SE) persisting after two antiseizure agents is called refractory (RSE), super-refractory status epilepticus (SRSE) defines SE continuing after general anaesthesia. Its prevalence and related clinical profiles have received limited attention, and most studies were restricted to intensive care facilities. We therefore aimed at describing RSE and SRSE frequencies and identifying associated clinical variables. METHODS: Between 2006 and 2015, consecutive adult SE episodes were prospectively recorded in a registry. Occurrence of RSE and SRSE and their relationship to clinical variables of interest, including outcome, were analysed. RESULTS: Of 804 SE episodes, 268 (33.3%) were RSE and 33 (4%) SRSE. Coma induction for SE treatment occurred in 79 (9.8%) episodes. Severe consciousness impairment (OR 1.67; 95% CI 1.24-2.46; P = 0.001), increasing age (OR 1.01, 95% CI 1.01-1.02), and lack of remote symptomatic SE aetiology (OR 0.48; 95% CI 0.32-0.72) were independently associated with RSE, while severe consciousness impairment (OR 4.26; 95% CI 1.44-12.60) and younger age (OR 0.96; 95% CI 0.95-0.99) correlated with SRSE; however, most SRSE episodes were not predicted by these variables. Mortality was 15.5% overall, higher in RSE (24.5%) and SRSE (37.9%) than in non-refractory SE (9.8%) (P < 0.001). SIGNIFICANCE: Super-refractory status epilepticus appears clearly less prevalent in this cohort than previously reported, probably as it is not restricted to intensive care unit. SRSE emerges in younger patients with marked consciousness impairment, pointing to the underlying severe clinical background, but these variables do not predict most SRSE developments. There is currently a knowledge gap for prediction of SRSE occurrence that needs to be filled.

PDGFRß(+) cells in human and experimental neuro-vascular dysplasia and seizures.

INTRODUCTION: Neuro-vascular rearrangement occurs in brain disorders, including epilepsy. Platelet-derived growth factor receptor beta (PDGFRß) is used as a marker of perivascular pericytes. Whether PDGFRß(+) cell reorganization occurs in regions of neuro-vascular dysplasia associated with seizures is unknown. METHODS: We used brain specimens derived from epileptic subjects affected by intractable seizures associated with focal cortical dysplasia (FCD) or temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Tissues from cryptogenic epilepsy, non-sclerotic hippocampi or peritumoral were used for comparison. An in vivo rat model of neuro-vascular dysplasia was obtained by pre-natal exposure to methyl-axozy methanoic acid (MAM). Status epilepticus (SE) was induced in adult MAM rats by intraperitoneal pilocarpine. MAM tissues were also used to establish organotypic hippocampal cultures (OHC) to further assess pericytes positioning at the dysplastic microvasculature. PDGFRß and its colocalization with RECA-1 or CD34 were used to segregate perivascular pericytes. PDGFRß and NG2 or IBA1 colocalization were performed. Rat cortices and hippocampi were used for PDGFRß western blot analysis. RESULTS: Human FCD displayed the highest perivascular PDGFRß immunoreactivity, indicating pericytes, and presence of ramified PDGFRß(+) cells in the parenchyma and proximal to microvessels. Tissues deriving from human cryptogenic epilepsy displayed a similar pattern of immunoreactivity, although to a lesser extent compared to FCD. In TLE-HS, CD34 vascular proliferation was paralleled by increased perivascular PDGFRß(+) pericytes, as compared to non-HS. Parenchymal PDGFRß immunoreactivity co-localized with NG2 but was distinct from IBA1(+) microglia. In MAM rats, we found pericyte-vascular changes in regions characterized by neuronal heterotopias. PDGFRß immunoreactivity was differentially distributed in the heterotopic and adjacent normal CA1 region. The use of MAM OHC revealed microvascular-pericyte dysplasia at the capillary tree lining the dentate gyrus (DG) molecular layer as compared to control OHC. Severe SE induced PDGFRß(+) immunoreactivity mostly in the CA1 region of MAM rats. CONCLUSION: Our descriptive study points to microvascular-pericyte changes in the epileptic pathology. The possible link between PDGFRß(+) cells, neuro-vascular dysplasia and remodeling during seizures is discussed.

Effect of status epilepticus and antiepileptic drugs on CYP2E1 brain expression.

P450 metabolic enzymes are expressed in the human and rodent brain. Recent data support their involvement in the pathophysiology of epilepsy. However, the determinants of metabolic enzyme expression in the epileptic brain are unclear. We tested the hypothesis that status epilepticus (SE) or exposure to phenytoin or phenobarbital affects brain expression of the metabolic enzyme CYP2E1. SE was induced in C57BL/6J mice by systemic kainic acid. Brain CYP2E1 expression was evaluated 18-24h after severe SE by immunohistochemistry. Co-localization with neuronal nuclei (NEUN), glial fibrillary acidic protein (GFAP) and CD31 was determined by confocal microscopy. The effect of phenytoin, carbamazepine and phenobarbital on CYP2E1 expression was evaluated in vivo or by using organotypic hippocampal cultures in vitro. CYP2E1 expression was investigated in brain resections from a cohort of drug-resistant epileptic brain resections and human endothelial cultures (EPI-EC). Immunohistochemistry showed an increase of CYP2E1 expression limited to hippocampal CA2/3 and hilar neurons after severe SE in mice. CYP2E1 expression was also observed at the astrocyte-vascular interface. Analysis of human brain specimens revealed CYP2E1 expression in neurons and vascular endothelial cells (EC). CYP2E1 was expressed in cultured human EC and over-expressed by EPI-EC. When analyzing the effect of drug exposure on CYP2E1 expression we found that, in vivo or in vitro, ethanol increased CYP2E1 levels in the brain and liver. Treatment with phenytoin induced localized CYP2E1 expression in the brain whereas no significant effects were exerted by carbamazepine or phenobarbital. Our data indicate that the effect of acute SE on brain CYP2E1 expression is localized and cell specific. Exposure to selected anti-epileptic drugs could play a role in determining CYP2E1 brain expression. Additional investigation is required to fully reproduce the culprits of P450 enzyme expression as observed in the human epileptic brain.

Use of a high-risk human papillomavirus DNA test as the primary test in a cervical cancer screening programme: a population-based cohort study.

OBJECTIVE: To present the results of the first 2 years of a human papillomavirus (HPV) test-based screening programme outside the research context. DESIGN: Population-based cohort study. SETTING: A cervical service screening programme in Italy. POPULATION: Women aged 25-64 years invited to screening from April 2009 to April 2011. METHODS: Eligible women were invited to undergo an HPV test: those with a negative HPV test went on to the next screening episode; those with a positive HPV went on to triage with a Pap smear. Women with positive cytology (i.e. positive for atypical squamous cells of undetermined significance or worse, ASC-US+) were referred to colposcopy, whereas those with negative cytology were referred to repeat HPV testing 1 year later. MAIN OUTCOME MEASURES: Participation rate, positivity at HPV and at triage, referral rate to colposcopy, positive predictive value for cervical intraepithelial neoplasia grade 2+ (CIN2+) at colposcopy, and detection rate for CIN2+. RESULTS: Participation increased compared with the previous Pap programme (60.6 versus 43.9%). The HPV positivity rate was 7.0; 39.6% of Pap smears were scored as positive, and therefore 2.8% of the women screened were referred for immediate colposcopy. The compliance of women who scored positive for HPV and negative for Pap for repeat HPV testing at 12 months was 78.6%, and the HPV positivity rate was 56.6%. The overall referral rate to colposcopy was 4.6%. The overall detection rate for CIN2+ was 4.5 versus 1.5% of the Pap programme (25-34 years, 8.2%; 35+ years, 3.6%). CONCLUSIONS: Compared with the traditional Pap test, the HPV programme recorded a higher response to invitation and an increased DR for CIN2+. The most critical aspects were the reading of cytology in women that were positive for HPV and the increased workload at colposcopy.

Vasectomy within the public health services in Campinas, São Paulo, Brazil.

OBJECTIVE: To describe some of the characteristics of men who underwent a vasectomy in the public health network of Campinas, São Paulo, Brazil. METHODS: A descriptive study including 202 men randomly selected from a list of all the men vasectomized between 1998 and 2004 in the public health network. RESULTS: Most of the men were 30 years of age or older when vasectomized, had completed elementary school and had two or more children of both sexes. Most of the men came from the lowest income segment of the population: 47.6% in 1998-1999 and 61.3% in 2003-2004. Although the men knew various contraceptive methods, 51.2% reported that their partners were using combined oral contraceptives at the time of surgery. Most men initially sought information on vasectomy at health-care clinics where care was provided by a multidisciplinary team; most received counselling, however, 47.9% of the men waited more than 4 months for the vasectomy. CONCLUSIONS: The profile of the vasectomized men in this study appears to indicate that the low-income population from Campinas, São Paulo, Brazil has access to vasectomy; however, the waiting time for vasectomy reveals that difficulties exist in obtaining this contraceptive method in the public health service.

Transporters in drug-refractory epilepsy: clinical significance.

Drug resistance remains an unmet challenge in a variety of neurological disorders, but epilepsy is probably the refractory disease that has received most experimental, preclinical, and therapeutic attention. Although resective surgery continues to improve our ability to provide seizure relief, new discoveries have potential as alternative therapeutic approaches to multiple drug resistance. As discussed here, the field is replete with controversies and false starts, in particular as it concerns the existence of genetic predisposition to inadequate pharmacological seizure control.

Fertility in a i(Xq) Klinefelter patient: importance of XIST expression level determined by qRT-PCR in ruling out Klinefelter cryptic mosaicism as cause of oligozoospermia.

The presence of an isochromosome Xq in Klinefelter syndrome (KS) is an apparently rare condition. In all cases reported so far, patients showed the classic phenotype. We here describe a case of isochromosome Xq [47,X,i(Xq),Y] in a non-mosaic KS patient. The patient exhibited a normal androgenized phenotype, normal testes and normal cognitive abilities. Semen analysis revealed a medium oligozoospermia (5 x 10(6) spermatozoa/ml). After the patient underwent intracytoplasmic sperm injection, he generated two cytogenetically healthy normal females. Fluorescence in situ hybridization analysis showed the presence of a dicentric Xq chromosome that did not show the presence of residual Xp arm up to the 57,820,478 bp position (Xp 1.1) of X chromosome sequence. Preferential inactivation of Xq isochromosome was demonstrated by bromodeoxyuridine replication analysis and transcriptional silencing by DNA methylation at the HUMARA locus. Furthermore, we demonstrated by quantitative RT-PCR an active XIST RNA expression in blood lymphocytes from Klinefelter patients, comparable to that observed in control females and over 30,000-fold greater than in control males. In conclusion, this qRT-PCR approach could be useful for screening of prepuberty males and for diagnosis or exclusion of cryptic Klinefelter mosaics.

Combined effects of prenatal inhibition of vasculogenesis and neurogenesis on rat brain development.

Malformations of cortical development (MCD) are one of the most common causes of neurological disabilities including autism and epilepsy. To disrupt cortical formation, methylazoxymethanol (MAM) or thalidomide (THAL) has been used to affect neurogenesis or vasculogenesis. Although previous models of MCD have been useful, these models primarily attack a single aspect of cortical development. We hypothesized that simultaneous prenatal exposure to MAM or THAL will lead to the development of a novel and specific type of brain maldevelopment. Rats were prenatally exposed to MAM and THAL. At early postnatal days, brains displayed abnormal ventricular size and hemispheric asymmetry due to altered brain water homeostasis. The postnatal brain was also characterized by gliosis in regions of focal leakage of the blood brain barrier. These morphological abnormalities gradually disappeared at adult stages. Although the adult MAM-THAL rats showed normal cortical morphology, abnormal hippocampal connectivity and mossy fiber sprouting persisted well into adulthood.

Contraceptive methods with male participation: a perspective of Brazilian couples.

OBJECTIVE: To assess the perspectives of couples who requested vasectomy in a public health service on the use of male participation contraceptive methods available in Brazil: male condoms, natural family planning/calendar, coitus interruptus and vasectomy. METHODS: A qualitative study with semi-structured interviews was held with 20 couples who had requested vasectomy at the Human Reproduction Unit of the Universidade Estadual de Campinas, Brazil. Data analysis was carried out through thematic content analysis. FINDINGS: The couples did not, in general, know any effective contraceptive options for use by men and/or participating in their use, except for vasectomy. The few methods with male participation that they knew of were perceived to interfere in spontaneity and in pleasure of intercourse. Men accepted that condom use in extra-conjugal relations offered them protection from sexually transmitted diseases; that their wives might also participate in extra-marital relationships was not considered. DISCUSSION: The few contraceptive options with male participation lead to difficulty in sharing responsibilities between men and women. On the basis of perceived gender roles, women took the responsibility for contraception until the moment when the situation became untenable, and they faced the unavoidable necessity of sterilization. CONCLUSIONS: Specific actions are necessary for men to achieve integral participation in relation to reproductive sexual health. These include education and discussions on gender roles, leading to greater awareness in men of the realities of sexual and reproductive health.

Acute induction of epileptiform discharges by pilocarpine in the in vitro isolated guinea-pig brain requires enhancement of blood-brain barrier permeability.

Systemic application of the muscarinic agonist, pilocarpine, is commonly utilized to induce an acute status epilepticus that evolves into a chronic epileptic condition characterized by spontaneous seizures. Recent findings suggest that the status epilepticus induced by pilocarpine may be triggered by changes in the blood-brain barrier (BBB) permeability. We tested the role of the BBB in an acute pilocarpine model by using the in vitro model brain preparation and compared our finding with in vivo data. Arterial perfusion of the in vitro isolated guinea-pig brain with <1 mM pilocarpine did not cause epileptiform activity, but rather reduced synaptic transmission and induced steady fast (20-25 Hz) oscillatory activity in limbic cortices. These effects were reversibly blocked by co-perfusion of the muscarinic antagonist atropine sulfate (5 microM). Brain pilocarpine measurements in vivo and in vitro suggested modest BBB penetration. Pilocarpine induced epileptiform discharges only when perfused with compounds that enhance BBB permeability, such as bradykinin (n=2) or histamine (n=10). This pro-epileptic effect was abolished when the BBB-impermeable muscarinic antagonist atropine methyl bromide (5 microM) was co-perfused with histamine and pilocarpine. In the absence of BBB permeability enhancing drugs, pilocarpine induced epileptiform activity only after arterial perfusion at concentrations >10 mM. Ictal discharges correlated with a high intracerebral pilocarpine concentration measured by high pressure liquid chromatography. We propose that acute epileptiform discharges induced by pilocarpine treatment in the in vitro isolated brain preparation are mediated by a dose-dependent, atropine-sensitive muscarinic effect promoted by an increase in BBB permeability. Pilocarpine accumulation secondary to BBB permeability changes may contribute to in vivo ictogenesis in the pilocarpine epilepsy model.

Prenatal exposure to thalidomide, altered vasculogenesis, and CNS malformations.

Malformations of cortical development (MCD) result from abnormal neuronal positioning during corticogenesis. MCD are believed to be the morphological and perhaps physiological bases of several neurological diseases, spanning from mental retardation to autism and epilepsy. In view of the fact that during development, an appropriate blood supply is necessary to drive organogenesis in other organs, we hypothesized that vasculogenesis plays an important role in brain development and that E15 exposure in rats to the angiogenesis inhibitor thalidomide would cause postnatal MCD. Our results demonstrate that thalidomide inhibits angiogenesis in vitro at concentrations that result in significant morphological alterations in cortical and hippocampal regions of rats prenatally exposed to this vasculotoxin. Abnormal neuronal development was associated with vascular malformations and a leaky blood-brain barrier. Protein extravasation and uptake of fluorescent albumin by neurons, but not glia, was commonly associated with abnormal cortical development. Neuronal hyperexcitability was also a hallmark of these abnormal cortical regions. Our results suggest that prenatal vasculogenesis is required to support normal neuronal migration and maturation. Altering this process leads to failure of normal cerebrovascular development and may have a profound implication for CNS maturation.

Teratogenic effects of lamotrigine on rat fetal brain: a morphometric study.

A study of the teratogenic activity of an antiepileptic drug - lamotrigine - was carried out in the brain of fetuses of rats who had received the drug. The dosage levels studied corresponded to four times the median effective dose (ED50) in rats. The drug was administered during the organogenesis period. Rats were sacrificed one day prior to term and fetuses were macroscopically examined, weighted and cephalic segments sectioned (Wilson technique), for histological study by stereological analysis, using Merz's grid for drawing and point counts. Cortex, subcortex, ependyma and lateral ventricles were analyzed. The same methodology was applied to the control group; data were compared with by the non-parametric Mann-Whitney statistical analysis test. Results showed that fetuses of the experimental group had reduced body weight at birth, increased volume and diameter of the cerebral structure, increased density of the subcortical layer, and ventricle dilatation. Possible mechanisms of this teratogenicity were discussed.

[Aicardi syndrome: case report]. / Síndrome de Aicardi: relato de caso.

We report a case of Aicardi syndrome in a female child with 2 month old, ocular abnormalities "chorioretinal lacunae", flexion in spasms, hipsarrhythmic "split brain", callosal agenesis referred to Service of Neuropediatric and Neurophysiology of Base Hospital of São José do Rio Preto, SP, Brazil.

Fluoxetine in the treatment of Huntington's disease.

RATIONALE: Recent reports suggest the possible efficacy of selective serotonin reuptake inhibitors (SSRIs) in treating particular symptoms of Huntington's disease (HD), such as aggressiveness and agitation. However, predictive features to identify HD subjects who may benefit from this treatment have not been established. OBJECTIVES: Two individuals from a large HD pedigree with a very high prevalence of obsessive-compulsive disorder (OCD) have been treated with fluoxetine, an SSRI. We aimed at testing whether the co-occurrence of the two disorders in this pedigree might have some underlying pathogenic similarities, maybe also resulting in a good response of HD symptoms to the anti-obsessional drug fluoxetine. METHODS: Each patient was evaluated, started on fluoxetine treatment, and then reassessed monthly with: (a) the HD motor rating scale, to rate the impairment of movement, and (b) the mini mental state examination, for a cognitive ascertainment. They had a complete psychiatric and neurologic examination as well. RESULTS: Both subjects showed an excellent response to fluoxetine. One patient exhibited improvement of the motor and behavioral components of the disorder, while the other improved also in the cognitive domain of HD. The best response was shown by the individual suffering from OCD in her youth. The amelioration in these two patients has been maintained for 2 and 6 years, respectively, whereas the course of HD is that of a progressive deterioration. CONCLUSIONS: Firm conclusions to explain these results cannot be drawn. However, a hypothetical involvement of the serotonergic system, suggested by the excess of OCD in the pedigree, seems supported by the response of these two individuals to fluoxetine. It may be worth further exploring the value of the psychiatric picture in selecting the appropriate treatment for at least some cases of HD.

The effect upon the human vaginal histology of the long-term use of the injectable contraceptive Depo-Provera.

The objective of the study was to evaluate the effect of long-term use of the injectable contraceptive depot medroxyprogesterone acetate (DMPA) on human vaginal histology. Twenty premenopausal women currently using DMPA as a contraceptive method for two and three years were compared with 20 regularly menstruating women, who never used Depo-Provera and/or other kind of hormonal contraceptive in the last 6 months prior to the study. Subjects and controls were matched by age (+/-1 year), body mass index (kg/m2) (+/-1.0), number of pregnancies (+/-1), age at first intercourse (+/-1 year), years of sexual activity (+/-1 year), and number of partners during their life (+/-1). Vaginal biopsies were performed in users at 90+/-7 days after the last injection and in nonusers at day 20-25 of the menstrual cycle. In addition, at the day of the biopsy a blood sample was collected to measure estradiol (in all women) and DMPA in users. The level of serum estradiol was significant lower in Depo-Provera users than in controls (p < 0.001). The thickness of the vaginal epithelium was not smaller among DMPA users than among controls, the mean count of Langerhans cells per mm of epithelium were almost identical in both groups, and no significant differences were found on the vaginal maturation indices. In conclusion, the use of Depo-Provera between two and three years did not affect vaginal thinning of the epithelium, Langerhans cell count or maturation index.

[Epileptic syndromes and seizures and their relationship with work: a prospective ambulatory study in 412 epileptic patients]. / As síndromes e crises epilépticas e suas relações com trabalho: estudo prospectivo ambulatorial de 412 pacientes.

This study aims to evaluate, prospectively, the epileptic syndromes and seizures types upon work based on a sample of 412 out-patients from Hospital de Base, São José do Rio Preto, SP, Brazil. It was observed that the epileptic syndromes were significant in relation to the patients' labor skills (p= 0.001): the idiopathic syndromes showed less prejudiced, while the symptomatic was more. The seizures types also had some influence in relation to the patients' labor skills (p=0.016): the generalized non-convulsive seizures had no involvement; the simple partial and the non-classified had moderately involvement; and the simple partial seizures evolving to complex and tonic-clonic generalized were the seizures which mostly have taken the patients away from work. The seizure severity was also analyzed.

Huntington's disease and its association with psychopathology.

We review several aspects of Huntington's disease (HD), with a special focus on the psychopathological manifestations often identified in patients with this disorder. We discuss the evidence for a higher-than-average prevalence of psychosis, depression, and obsessive-compulsive disorder (OCD) in individuals with HD or at risk for the illness and analyze the possible significance of these findings. Particular emphasis is placed on OCD, in view of the neuroanatomical impairment that this condition shares with HD, the symptomatic similarities between these disorders, and recent findings of an excess of OCD in HD-affected families. We hypothesize that precise characterization of the psychiatric status of some HD patients showing psychopathological manifestations and their families might help to distinguish different clinical subtypes of the disorder. This approach could hold promise in improving the management of HD in the future.

Association of obsessive-compulsive disorder and pathological gambling with Huntington's disease in an Italian pedigree: possible association with Huntington's disease mutation.

This paper describes a pedigree with Huntington's disease (HD), in which three cases of obsessive-compulsive disorder (OCD) and two cases of pathological gambling (PG) were identified. The mutation analysis of the HD gene was carried out in the examined individuals who were at risk for HD. In fact, OCD and PG only occurred in carriers of the HD expansion. The possible implications of this finding are discussed.