[The heterogeneity of blood flow on magnetic resonance imaging: a biomarker for grading cerebral astrocytomas]. / La heterogeneidad del flujo sanguíneo en resonancia magnética, biomarcador para clasificar por grados los astrocitomas cerebrales.

OBJECTIVES: To study whether the histograms of quantitative parameters of perfusion in MRI obtained from tumor volume and peritumor volume make it possible to grade astrocytomas in vivo. MATERIAL AND METHODS: We included 61 patients with histological diagnoses of grade II, III, or IV astrocytomas who underwent T2*-weighted perfusion MRI after intravenous contrast agent injection. We manually selected the tumor volume and peritumor volume and quantified the following perfusion parameters on a voxel-by-voxel basis: blood volume (BV), blood flow (BF), mean transit time (TTM), transfer constant (K(trans)), washout coefficient, interstitial volume, and vascular volume. For each volume, we obtained the corresponding histogram with its mean, standard deviation, and kurtosis (using the standard deviation and kurtosis as measures of heterogeneity) and we compared the differences in each parameter between different grades of tumor. We also calculated the mean and standard deviation of the highest 10% of values. Finally, we performed a multiparametric discriminant analysis to improve the classification. RESULTS: For tumor volume, we found statistically significant differences among the three grades of tumor for the means and standard deviations of BV, BF, and K(trans), both for the entire distribution and for the highest 10% of values. For the peritumor volume, we found no significant differences for any parameters. The discriminant analysis improved the classification slightly. CONCLUSIONS: The quantification of the volume parameters of the entire region of the tumor with BV, BF, and K(trans) is useful for grading astrocytomas. The heterogeneity represented by the standard deviation of BF is the most reliable diagnostic parameter for distinguishing between low grade and high grade lesions.

Tubed myocutaneous pectoralis major flap: A rescue option in esophagus reconstruction.

This article presents a complex case of total pharyngoesophageal defect reconstruction in a patient with recurrent supraglottic squamous cell carcinoma. After failed attempts with free microsurgical techniques due to complications, a tubed myocutaneous pectoralis major flap was successfully employed. The procedure aimed to achieve alimentary continuity, speech, and swallowing functionality. Despite initial challenges and concerns about stenosis, the reconstruction of a total pharyngolarygectomy defect resulted in a good functional outcome, although minor deficits in neck mobility and aesthetic donor zone deformities were noted. The case highlights the broad armamentarium of reconstruction techniques that plastic and aesthetic surgery teams must be trained, namely the myocutaneous pectoralis major flap which in selected cases can be the end solution.

Systematic resequencing of X-chromosome synaptic genes in autism spectrum disorder and schizophrenia.

Autism spectrum disorder (ASD) and schizophrenia (SCZ) are two common neurodevelopmental syndromes that result from the combined effects of environmental and genetic factors. We set out to test the hypothesis that rare variants in many different genes, including de novo variants, could predispose to these conditions in a fraction of cases. In addition, for both disorders, males are either more significantly or more severely affected than females, which may be explained in part by X-linked genetic factors. Therefore, we directly sequenced 111 X-linked synaptic genes in individuals with ASD (n = 142; 122 males and 20 females) or SCZ (n = 143; 95 males and 48 females). We identified >200 non-synonymous variants, with an excess of rare damaging variants, which suggest the presence of disease-causing mutations. Truncating mutations in genes encoding the calcium-related protein IL1RAPL1 (already described in Piton et al. Hum Mol Genet 2008) and the monoamine degradation enzyme monoamine oxidase B were found in ASD and SCZ, respectively. Moreover, several promising non-synonymous rare variants were identified in genes encoding proteins involved in regulation of neurite outgrowth and other various synaptic functions (MECP2, TM4SF2/TSPAN7, PPP1R3F, PSMD10, MCF2, SLITRK2, GPRASP2, and OPHN1).

In vitro assessment of gentian violet anti- candida activity.

BACKGROUND/AIM: Recurrent vulvovaginal candidosis (RVVC) needs alternative therapeutic approaches. Gentian violet (GeV) has been traditionally used to treat mucocutaneous candidosis. The aim of the present study was to evaluate the in vitro activity of GeV against Candida spp. and contribute to clarify the mechanism of action, supporting its clinical therapeutic use. METHODS: Seventeen clinical Candida isolates from RVVC and one C. albicans type collection (ATCC 10231) were studied; the antifungal activity of GeV was evaluated according to the CLSI M27-A3 protocol. To elucidate its mechanism of action, cells were stained with propidium iodide and afterwards analyzed by flow cytometer. RESULTS: GeV showed a fungicidal activity against most Candida spp. C. albicans and C. tropicalis were the most susceptible species. Minimal lethal concentrations were similar to minimal inhibitory concentrations for most tested strains. The fungicidal effect was not related to a primary lesion of the cytoplasmic membrane. CONCLUSION: In accordance with our findings, GeV is a valuable potent fungicidal drug to be used topically, isolated or in combination with oral antifungal drugs, particularly in RVVC cases.

Mutations in the APC tumour suppressor gene cause chromosomal instability.

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Anti-Candida activity of a chitosan hydrogel: mechanism of action and cytotoxicity profile.

Candida spp. are common causative agents of mucocutaneous infections. New therapeutic antifungal drugs are needed to treat chronic disease as these are frequently clinically resistant to azols. Chitosan, among other possible vehicles for active compounds, shows an added value as it appears to have intrinsic antimicrobial properties. The aim of the present study was to evaluate the anti-Candida activity of a medium-molecular-weight chitosan hydrogel (CH), to clarify its possible mechanism of action and to evaluate its cytotoxicity on human fibroblasts. CH antifungal activity was assessed according to CLSI reference M27-A3 protocol; its mechanism of action was investigated by flow cytometry, and its cytotoxicity was studied by MTT assay. CH demonstrated a full inhibition of C. tropicalis, C. krusei, C. guilliermondii and C. parapsilosis growth while impairing C. albicans and C. glabrata viability. Flow cytometry tests showed that CH acts by inducing primary lesion of the cytoplasmic membrane. However, CH showed no cytotoxic effect upon human fibroblasts cells. Resistant strains will require new therapeutic approaches. Chitosan being a good carrier and having itself anti-Candida activity seems to be a promising vehicle to be used for the treatment of mucocutaneous candidosis.

Offloading memory leaves us vulnerable to memory manipulation.

We often offload memory demands onto external artefacts (e.g., smartphones). While this practice allows us to subvert the limitations of our biological memory, storing memories externally exposes them to manipulation. To examine the impact of such manipulation, we report three experiments, two of which were pre-registered. Individuals performed a memory task where they could offload to-be-recalled information to an external store and on a critical trial, we surreptitiously manipulated the information in that store. Results demonstrated that individuals rarely noticed this manipulation. In addition, when individuals had information inserted into their external memory stores, they often encoded it into their biological memory, thereby leading to the creation of a false memory. The reported results highlight one of the cognitive consequences of offloading our memory to external artefacts.

[Lethal pulmonary involvement of neutrophilic dermatosis following erythropoietin therapy]. / Extension pulmonaire d'une dermatose neutrophilique rapidement létale dans les suites d'un traitement par érythropoïétine.

BACKGROUND: Neutrophilic disease is characterized by aseptic visceral infiltration by normal polymorphonuclear leukocytes that can occur in any organ. Association with an underlying systemic disease, particularly haematological malignancy or inflammatory bowel disease, is frequent. This may produce a multisystem disorder, but diagnosis is usually based on skin lesions because of their clinical and histological accessibility. Pulmonary manifestations are the most common extracutaneous symptoms but may be misdiagnosed, as in our case report. CASE REPORT: A 77-year-old woman with IgA myeloma presented with an inflammatory bullous plaque of the leg coupled with fever lasting one week. The clinical and histological examinations were evocative of a neutrophilic dermatosis such as Sweet's syndrome. Significant improvement was initially obtained with systemic corticosteroids and colchicine. The course became complicated by necrotic neutrophilic papulopustular lesions of the upper limbs and pulmonary manifestations, with fever and decline in overall condition occurring the day after administration of erythropoietin. A hypothesis of septic aetiology prompted antibiotic and antifungal therapy, which remained ineffective. The patient died the day after the second erythropoietin injection. DISCUSSION: This case involved late identification of the aseptic neutrophilic aetiology of pulmonary manifestations. Several factors favouring their appearance and the fatal outcome may be suggested: the existence of a myeloma, association with myelodysplastic syndrome and the possible iatrogenic action of erythropoietin. To the best of our knowledge, this is the first reported case of extracutaneous neutrophilic infiltrate occurring in a patient treated with this haematopoietic hormone.

Bacteriocin production of the probiotic Lactobacillus acidophilus KS400.

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.

Outcomes of phacoemulsification in patients with uveitis at a tertiary center in São Paulo, Brazil: a review of cases from 2007 to 2012.

PURPOSE:: To evaluate the outcomes of phacoemulsification with intraocular lens (IOL) implantation in eyes with uveitis. METHODS:: Consecutive patients with uveitis and cataracts who had phacoemulsification and acrylic IOL implantation during 2007-2012 were evaluated for visual outcomes, etiology, and complications. Inflammation was controlled for at least 3 months before surgery, and oral prednisone (0.5 mg/kg/day) was administered to patients with non-infectious uveitis. RESULTS:: This study investigated 45 eyes in 38 patients with a mean age of 52 ± 12.5 years. The most common etiologies among non-infectious causes (n=32; 73.3%) were Vogt-Koyanagi-Harada disease (n=9), Fuchs heterochromic iridocyclitis (n=4), and sympathetic ophthalmia (n=3). Four cases were idiopathic. Among infectious cases (n=13; 28.9%), the most common causes were toxoplasmosis (n=6) and presumed ocular tuberculosis (n=4). An acrylic IOL was implanted in-the-bag in all cases. After 1-year follow-up, an improvement in visual acuity of two or more lines was observed in 38 eyes (84.4%), and 28 eyes (62.2%) achieved a postoperative visual acuity of ≥0.5. Posterior capsule opacification was observed in 10 eyes (22.2%). Persistent postoperative inflammation (of >6 months) was observed in seven eyes (15.5%) and recurrence occurred in four eyes (8.8%). IOL was explanted in one eye. Intraocular hypertension was observed in six eyes (13.3%). CONCLUSIONS:: Phacoemulsification with IOL implantation improved vision in most patients with coexisting cataracts and uveitis. Good preoperative and postoperative control of inflammation plays an important role in achieving favorable visual outcomes. Furthermore, the final visual outcome depends on the posterior segment status.

Real-time myocardial perfusion imaging for pharmacologic stress testing: added value to single photon emission computed tomography.

BACKGROUND: Little is known about the incremental value of real-time myocardial contrast echocardiography (MCE) as an adjunct to pharmacologic stress testing. This study was performed to evaluate the diagnostic value of MCE to detect abnormal myocardial perfusion by technetium Tc 99m sestamibi-single photon emission computed tomography (SPECT) and anatomically significant coronary artery disease (CAD) by angiography. METHODS: Myocardial contrast echocardiography was performed at rest and during vasodilator stress in consecutive patients (N = 120) undergoing SPECT imaging for known or suspected CAD. Myocardial opacification, wall motion, and tracer uptake were visually analyzed in 12 myocardial segments by 2 pairs of blinded observers. Concordance between the 2 methods was assessed using the kappa statistic. RESULTS: Of 1356 segments, 1025 (76%) were interpretable by MCE, wall motion, and SPECT. Sensitivity of wall motion was 75%, specificity 83%, and accuracy 81% for detecting abnormal myocardial perfusion by SPECT (kappa = 0.53). Myocardial contrast echocardiography and wall motion together yielded significantly higher sensitivity (85% vs 74%, P < .05), specificity of 83%, and accuracy of 85% (kappa = 0.64) for the detection of abnormal myocardial perfusion. In 89 patients who underwent coronary angiography, MCE and wall motion together yielded higher sensitivity (83% vs 64%, P < .05) and accuracy (77% vs 68%, P < .05) but similar specificity (72%) compared with SPECT for the detection of high-grade, stenotic (> or = 75%) coronary lesions. CONCLUSION: Assessment of myocardial perfusion adds value to conventional stress echocardiography by increasing its sensitivity for the detection of functionally abnormal myocardial perfusion. Myocardial contrast echocardiography and wall motion together provide higher sensitivity and accuracy for detection of CAD compared with SPECT.

Microbiological quality control of non-sterile compounded medicines prepared in a Portuguese hospital centre.

OBJECTIVES: This work aimed to evaluate the quality of non-sterile formulations compounded at Centro Hospitalar Cova da Beira (Covilhã, Portugal) immediately after preparation and up to the defined 'beyond-use date'. METHODS: Microbiological quality control tests were performed in accordance with monograph 5.1.4 of the European Pharmacopoeia 8.0. Samples of compounded products were collected from January to December 2014 after preparation and were analysed immediately and reanalysed after storage under the established conditions, for each preparation. RESULTS: In the test period, 392 preparations were analysed, corresponding to 24 different formulations (8 intermediate preparations, 11 oral solutions/suspensions and 5 topical preparations). All preparations were in accordance with the pharmacopoeia specifications immediately after preparation. However, for the formulations 'prednisolone oral solution (5 mg/mL)' and 'nitroglycerine and cinchocaine ointment (0.25%/0.5%)', the microbial counts of some batches exceeded the defined limits after storage up to the beyond-use date. CONCLUSIONS: These results show that the compounding practices implemented at this pharmacy department are able to ensure the microbiological quality of compounded products. This microbiological quality control methodology also allowed identification of the need to replace formulations shown not to be stable throughout the storage period. On the basis of these results, a monthly routine of microbiological control of a random sample of compounded medicines was established in order to ensure their quality and safety for use.

Radiation induces different changes in expression profiles of normal rectal tissue compared with rectal carcinoma.

PURPOSE: Radiotherapy is a very effective adjuvant treatment for rectal cancer with little side effects. Its killing effect on tumor cells seems to be more profound than the effect on normal tissue. The molecular events caused by irradiation are mainly analyzed in in vitro and animal models; investigations on human material are rare. In the current study, we analyzed the effects of irradiation on gene expression in normal and tumor tissue of rectal cancer patients. METHODS AND MATERIALS: Normal and carcinoma tissue of patients from a randomized clinical trial of the benefits of preoperative radiotherapy were analyzed using the Affymetrix Human Cancer Gene Chip. Preoperative radiotherapy was given within 5 days prior to surgery. Results for normal tissue and tumor were compared to investigate the radiation-related differences between normal and tumor cells. We clustered the differentially expressed genes based on their functional annotation. Results were compared with immunohistochemical and literature data. RESULTS: The majority of the investigated cancer-related genes remained unchanged by irradiation (92% in tumor tissue and 93% in normal tissue). The differentially expressed genes varied between tumor and normal tissue except for maspin and IL-8. Both in tumor and normal tissue, differentially expressed genes were present related to cell signaling and cycle control, apoptosis and cell survival and tissue response and repair. However, the spectrum of affected genes was totally different. CONCLUSION: Pre-existing differences in gene expression between normal tissue and tumor tissue might explain the differences in their responses to radiation. This change in response may explain the clinical beneficial effect of radiotherapy on tumor cells (low local recurrence rate) and the less severe effects on normal tissue (minor side effects).

Estimation of the collective ionizing dose in the Portuguese population for the years 2011 and 2012, due to nuclear medicine exams.

OBJECTIVES: In 2009-2010 a Portuguese consortium was created to implement the methodologies proposed by the Dose Datamed II (DDM2) project, aiming to collect data from diagnostic X-ray and nuclear medicine (NM) procedures, in order to determine the most frequently prescribed exams and the associated ionizing radiation doses for the Portuguese population. The current study is the continuation of this work, although it focuses only on NM exams for the years 2011 and 2012. MATERIAL AND METHODS: The annual frequency of each of the 28 selected NM exams and the average administered activity per procedure was obtained by means of a nationwide survey sent to the 35 NM centres in Portugal. RESULTS: The results show a reduction of the number of cardiac exams performed in the last two years compared with 2010, leading to a reduction of the annual average effective dose of Portuguese population due to NM exams from 0.08 mSv ± 0.017 mSv/caput to 0.059 ± 0.011 mSv/caput in 2011 and 0.054 ± 0.011 mSv/caput in 2012. Portuguese total annual average collective effective dose due to medical procedures was estimated to be 625.6 ± 110.9 manSv in 2011 and 565.1 ± 117.3 manSv in 2012, a reduction in comparison with 2010 (840.3 ± 183.8 manSv). CONCLUSIONS: The most frequent exams and the ones that contributed the most for total population dose were the cardiac and bone exams, although a decrease observed in 2011 and in 2012 was verified. The authors intend to perform this study periodically to identify trends in the annual Portuguese average effective dose and to help to raise awareness about the potential dose optimization.

Expression of prolactin and its receptor in the baboon uterus during the menstrual cycle and pregnancy.

PRL is known to be a major secretory product of the human decidua. However, the physiological role of decidual PRL during the menstrual cycle and pregnancy has not been fully defined, primarily due to the lack of an appropriate nonhuman primate model for in vivo studies. Therefore, this initial study examined the expression of PRL and its receptor in the baboon uterus during the cycle and pregnancy. PRL and PRL receptor messenger ribonucleic acid expression were detected by semiquantitative RT-PCR, and protein was localized by immunocytochemistry. PRL was shown to be expressed in myometrial smooth muscle during the follicular phase by both RT-PCR and immunocytochemistry. Expression of PRL messenger ribonucleic acid and protein was first observed in the epithelial cells of the deep basal glands during the late luteal phase. With the onset of pregnancy, PRL expression increased steadily and was evident primarily in the decidual tissue. In contrast to PRL, its receptor was expressed at constant levels in both the myometrium and endometrium during the cycle. An increase in receptor expression was evident in both the decidua and placenta throughout pregnancy. In summary, these results demonstrate that the baboon uterus is a site of both PRL production and action during the cycle and pregnancy. These studies establish the baboon as a nonhuman primate model to investigate the potential roles of PRL in implantation and maintenance of pregnancy.

Detection of mutations in mismatch repair genes in Portuguese families with hereditary non-polyposis colorectal cancer (HNPCC) by a multi-method approach.

Mutation searching was performed in the hMSH2 and hMLH1 genes in 20 Portuguese families representing 124 registered affected individuals. Of the 20, 16 fulfilled the classic 'Amsterdam' criteria for HNPCC, whereas the remaining four families satisfied a modified set of criteria. These criteria required a CRC diagnosed before age 50 years and cancers diagnosed in two other relatives within the HNPCC spectrum. A multi-method approach was performed using the protein truncation test (PTT), single strand conformation polymorphism (SSCP) with two different sets of conditions, heteroduplex analysis (HA) and denaturing gradient gel electrophoresis (DGGE). Putative phenotype-genotype correlations were also explored. Ten different germline mutations were identified. Six of these were found in hMLH1 in seven families and four in hMSH2 in four families. SSCP and DGGE had the highest diagnostic yields with the percentage of variants detected above 67% and together HA and PTT had the lowest. No single technique detected all variants. Trends for the absence of extracolonic manifestations were observed in families carrying hMLH1 germline mutations (four of seven in hMLH1 vs one of four in hMSH2). Most of the families with rectal cancer were associated with hMLH1 (six of seven in hMLH1 vs two of four in hMSH2). A multi-technique approach is necessary to identify a high percentage of germline mutations. Seven novel mutations were found in this Portuguese population.

Study of three intragenic polymorphisms in the Machado-Joseph disease gene (MJD1) in relation to genetic instability of the (CAG)n tract.

Intergenerational instability is one of the most important features of the disease-associated trinucleotide expansions, leading to variation in size of the repeat among and within families, which manifests as variable age at onset and severity, and is probably the basis for the occurrence of anticipation. Several factors are known to affect the degree of instability, namely the type of repeated sequence, its initial size, the presence or absence of interruptions in the repetitive tract and the gender of the transmitting parent. A recent study demonstrated the effect of an intragenic polymorphism (C987GG/G987GG) in the Machado-Joseph disease causative gene, immediately downstream of the CAG repeat, on the intergenerational instability of the expanded repeat. Surprisingly, there was an effect not only of the specific allele in cis to the disease chromosome, but also of the allele on the normal chromosome, suggesting the existence of an interaction between the normal and expanded alleles that affects the fidelity of replication of the (CAG)n tract. This effect could be a direct effect of the polymorphism studied or, alternatively, this polymorphism could be in disequilibrium with some other flanking sequence which affects the instability of the repetitive (CAG)n tract. In order to confirm the previous results in a different population and to distinguish between a direct and indirect effect of the CGG/GGG polymorphism, we typed 70 parent-progeny pairs for which the variation in the (CAG)n length in the MJD1 gene was known, for three intragenic polymorphisms: C987GG/G987GG and two additional, newly described ones, TAA1118/TAC1118 and A669TG/G669TG. We also typed a control population of 125 individuals for the A669TG/G669TG, C987GG/G987GG and TAA1118/TAC1118 polymorphisms, in an attempt to identify any association between haplotype and (CAG)n length in normal chromosomes, suggestive of an instability-predisposing effect of the repeat-flanking sequences, which could have led to the origin of the MJD mutation in the human population. We confirmed the effect of the C987GG/G987GG polymorphism on intergenerational instability when present in trans. Our results suggest that this effect is restricted to a small region of the gene, immediately downstream of the CAG repeat, which includes this particular nucleotide substitution and the stop codon of the MJD1 cDNA, and is not a more widespread chromosomal effect. The lack of a significant association of any specific intragenic haplotype with larger CAG repeats in normal chromosomes, together with the absence of an effect of the intragenic haplotype in cis on the intergenerational instability of the expanded (CAG)n in MJD families does not indicate the existence of an instability-predisposing haplotype.

Improvement in the molecular diagnosis of Machado-Joseph disease.

BACKGROUND: Direct detection of the gene mutation allows for the confirmation of the clinical diagnosis of Machado-Joseph disease (MJD), the most frequent cause of autosomal dominant spinocerebellar ataxia worldwide. OBJECTIVE: To address the main difficulties in our national MJD predictive testing program. The first was the emergence of intermediate alleles, for which it is not yet possible to determine whether they will cause disease. The second was the issue of homoallelism, ie, homozygosity for 2 normal alleles with exactly the same (CAG)(n) length, which occurs in about 10% of all test results. METHODS: A large pedigree with 1 affected patient carrying a 71 and a 51 CAG repeat and 2 asymptomatic relatives carrying the 51 CAG repeat and normal-size alleles underwent clinical and molecular studies. Intragenic haplotypes for these alleles were determined. A representative sample of the healthy population in the region was obtained to assess the distribution of the normal (CAG)(n) length. We established the genotype for 4 intragenic polymorphisms in the gene for MJD (MJD1) in 21 homoallelic individuals, to distinguish their 2 normal chromosomes. In addition, we developed a new Southern blot method to completely exclude cases of nonamplification of expanded alleles in the homoallelic individuals. RESULTS: The study of the family in which the 51 CAG repeat was found suggests that the allele is apparently not associated with disease. These intermediate alleles were not present in a large sample of the healthy population from the same region. Intragenic polymorphisms allowed distinction of the 2 different normal alleles in all cases of homoallelism. The absence of an expanded allele was also confirmed by Southern blot. CONCLUSIONS: We propose an improved protocol for molecular testing for MJD. These strategies, developed to overcome the practical difficulties mostly in the presymptomatic and prenatal diagnosis of MJD, should prove useful for other polyglutamine-related disorders.