Cláudia Vianna Maurer-Morelli

Prospective neuroimaging study in hereditary spastic paraplegia with thin corpus callosum.

Our objective was to estimate the frequency as well as to establish the clinical and neuroimaging profile of hereditary spastic paraplegia with thin corpus callosum (HSP‐TCC). HSP‐TCC was recognized as a specific clinical subtype of HSP and mapped to chromosome (ch) 15q13‐15 in Japanese families. It has been considered rare in western countries. We assessed 45 patients with autosomal recessive HSP from 20 different families in search of clinical and imaging criteria for the diagnosis of HSP‐TCC. In addition, HSP‐TCC patients underwent further neurological, imaging and genetic evaluation. MRI scans were performed in a 2T scanner and sagittal T1 weighted images used for semiautomated volumetric measurements of corpus callosum, cerebellum, and brain. In seven patients, a 2‐year follow‐up MRI scan was performed. We genotyped seven microsatellite markers flanking the 15q13‐15 candidate region and calculated two‐point and multipoint LOD scores (Z). We identified 13 patients from seven unrelated families with HSP‐TCC. MRI showed significant corpus callosum, cerebral and cerebellar volumetric reductions (P < 0.001, P = 0.03, and P = 0.01, respectively). In the prospective analysis, we found progressive corpus callosum atrophy (P = 0.04). Two‐point and multipoint LOD scores were significantly negative for markers genotyped on ch 15q. However, independent pedigree analysis did not yield significant results. HSP‐TCC was found in 35% of families with autosomal recessive HSP. MRI volumetry showed cerebral and cerebellar atrophy in association with progressive corpus callosum thinning. Genetic studies did not show evidence for linkage to ch 15q.

Downregulation of 14q32 microRNAs in Primary Human Desmoplastic Medulloblastoma

Medulloblastoma (MB) is one of the most common pediatric cancers, likely originating from abnormal development of cerebellar progenitor neurons. MicroRNA (miRNA) has been shown to play an important role in the development of the central nervous system. Microarray analysis was used to investigate miRNA expression in desmoplastic MB from patients diagnosed at a young age (1 or 2 years old). Normal fetal or newborn cerebellum was used as control. A total of 84 differentially expressed miRNAs (64 downregulated and 20 upregulated) were found. Most downregulated miRNAs (32/64) were found to belong to the cluster of miRNAs at the 14q32 locus, suggesting that this miRNA locus is regulated as a module in MB. Possible mechanisms of 14q32 miRNAs downregulation were investigated by the analysis of publicly available gene expression data sets. First, expression of estrogen-related receptor-γ (ESRRG), a reported positive transcriptional regulator of some 14q32 miRNAs, was found downregulated in desmoplastic MB. Second, expression of the parentally imprinted gene MEG3 was lower in MB in comparison to normal cerebellum, suggesting a possible epigenetic silencing of the 14q32 locus. miR-129-5p (11p11.2/7q32.1), miR-206 (6p12.2), and miR-323-3p (14q32.2), were chosen for functional studies in DAOY cells. Overexpression of miR-129-5p using mimics decreased DAOY proliferation. No effect was found with miR-206 or miR-323 mimics.

MRI and EEG as long-term seizure outcome predictors in familial mesial temporal lobe epilepsy

Objective: To evaluate the natural history and outcome predictors in familial mesial temporal lobe epilepsy (FMTLE). Methods: We conducted a longitudinal study of 103 individuals from 17 FMTLE families (mean follow-up: 7.6 years). We divided subjects into 3 groups: FMTLE (n = 53), unclassified seizure (n = 18), and asymptomatics (n = 32). We divided FMTLE patients into 3 subgroups: seizure-free (n = 19), infrequent (n = 17) seizures, and frequent (n = 17) seizures and further reclassified them into favorable and poor outcome. We defined hippocampal atrophy (HA) by visual MRI analysis and performed volumetry in those who had 2 MRIs. Results: FMTLE patients with infrequent seizures evolved to either frequent seizures (17.6%) or seizure freedom (23.5%). In the seizure-free group, most remained seizure-free and 21% developed infrequent seizures. All patients with frequent seizures remained in the same status or underwent surgery. Twelve percent of the asymptomatics and 22% of the unclassified-seizure group evolved to FMTLE with infrequent seizures. Predictive factors of poor outcome were presence of HA (p = 0.0192) and interictal epileptiform discharges (p = 0.0174). The relationship between initial precipitating incidents and clinical outcome was not significant although a tendency was observed (p = 0.055). Use of antiepileptic drugs and secondary generalized seizures during the patient’s lifetime did not predict poor outcome. We observed progression of HA only in the group with frequent seizures. Conclusion: Most patients with FMTLE continued in the same clinical status. However, patients with frequent seizures had progression of HA and none improved except those who underwent surgery. Interictal epileptiform discharges and HA predicted poorer outcome in FMTLE, and there was a tendency in favor of initial precipitating incidents as outcome predictors.

Fluoro-Jade, but not Fluoro-Jade B, stains non-degenerating cells in brain and retina of embryonic and neonatal rats.

Fluoro-Jade (FJ) and Fluoro-Jade B (FJB) are fluorescein derivatives currently used to stain brain cells under degeneration. In this study, we investigated the FJ staining of nondegenerating cells in embryonic and neonatal rat brain and retina. In embryonic rat brain (embryonic day 15; E15), very intense staining of cells was observed. The number of FJ-stained cells and the intensity of staining decreased with increasing in animal age, being almost absent by postnatal day 16 (P16). Only a few cells in neonatal rat brain were in the process of cell death, as verified by the TUNEL technique. The FJ-stained cells in neonatal brain were positive for the neuronal marker neuronal nuclei antigen (NeuN). In retina, FJ stained mainly cells from the ganglion cell layer at P2 and the neuroblastic layer at P2 and P6. In contrast to FJ, FJB did not stain nondegenerating cells in embryonic and neonatal rats. These results show that in addition to staining degenerating brain cells, FJ also stains nondegenerating central nervous system cells in embryonic and neonatal stages.

Investigation of genetic factors underlying typical orofacial clefts : mutational screening and copy number variation

Typical orofacial clefts (OFCs) comprise cleft lip, cleft palate and cleft lip and palate. The complex etiology has been postulated to involve chromosome rearrangements, gene mutations and environmental factors. A group of genes including IRF6, FOXE1, GLI2, MSX2, SKI, SATB2, MSX1 and FGF has been implicated in the etiology of OFCs. Recently, the role of the copy number variations (CNVs) has been studied in genetic defects and diseases. CNVs act by modifying gene expression, disrupting gene sequence or altering gene dosage. The aims of this study were to screen the above-mentioned genes and to investigate CNVs in patients with OFCs. The sample was composed of 23 unrelated individuals who were grouped according to phenotype (associated with other anomalies or isolated) and familial recurrence. New sequence variants in GLI2, MSX1 and FGF8 were detected in patients, but not in their parents, as well as in 200 control chromosomes, indicating that these were rare variants. CNV screening identified new genes that can influence OFC pathogenesis, particularly highlighting TCEB3 and KIF7, that could be further analyzed. The findings of the present study suggest that the mechanism underlying CNV associated with sequence variants may play a role in the etiology of OFC.

Preliminary Analysis of the Nonsynonymous Polymorphism rs17563 in BMP4 Gene in Brazilian Population Suggests Protection for Nonsyndromic Cleft Lip and Palate.

Cleft lip with or without palate (CL±P) is common congenital anomalies in humans. Experimental evidence has demonstrated that bone morphogenetic protein 4 gene (Bmp4) is involved in the etiology of CL±P in animal models. The nonsynonymous polymorphism rs17563 T>C (p.V152A) in the BMP4 gene has been associated to the risk of nonsyndromic CL±P in Chinese population and microforms from different ethnic backgrounds. The aim of this study was to investigate the role of BMP4 gene in CL±P in Brazilian sample using genetic association approach. Our sample was composed by 123 patients with nonsyndromic CL±P and 246 controls, in which absence of CL±P was confirmed in 3 generations. The rs17563 polymorphism was genotyped by PCR-RFLP technique. Logistic regression was performed to evaluate allele and genotype association. Our data showed statistical power to detect association (86.83%) in this sample. Logistic regression results showed significant association between C allele and CL±P (P = 0.00018, OR = 0.40, and 95% CI = 0.25–0.65), as well as CC genotype and CL±P (P = 0.00018, OR = 0.35, and 95% CI = 0.19–0.66). So, there is a strong association between nonsyndromic CL±P and BMP4 rs17563 polymorphism in our sample and the C allele had a protective effect against the occurrence of nonsyndromic CL±P.

Normal ATXN3 Allele but Not CHIP Polymorphisms Modulates Age at Onset in Machado–Joseph Disease

Background: Age at onset (AO) in Machado–Joseph disease (MJD) is closely associated with the length of the CAG repeat at the mutant ATXN3 allele, but there are other intervening factors. Experimental evidence indicates that the normal ATXN3 allele and the C-terminal heat shock protein 70 (Hsp70)-interacting protein (CHIP) may be genetic modifiers of AO in MJD. Methods: To investigate this hypothesis, we determined the length of normal and expanded CAG repeats at the ATXN3 gene in 210 unrelated patients with MJD. In addition, we genotyped five single nucleotide polymorphisms (SNPs) within the CHIP gene. We first compared the frequencies of the different genotypes in two subgroups of patients who were highly discordant for AO after correction for the length of the expanded CAG allele. The possible modifier effect of each gene was then evaluated in a stepwise multiple linear regression model. Results: AO was associated with the length of the expanded CAG allele (r2 = 0.596, p < 0.001). Frequencies of the normal CAG repeats at the ATXN3 gene and of CHIP polymorphisms did not differ significantly between groups with highly discordant ages at onset. However, addition of the normal allele improved the model fit for prediction of AO (r2 = 0.604, p = 0.014). Indeed, we found that the normal CAG allele at ATXN3 had a positive independent effect on AO. Conclusion: The normal CAG repeat at the ATXN3 gene has a small but significant influence on AO of MJD.

Insertional Translocation of 15q25-q26 into 11p13 and Duplication at 8p23.1 Characterized by High Resolution Arrays in a Boy With Congenital Malformations and Aniridia

We report on a boy presenting submucous cleft palate, hydronephrosis, ventriculoseptal defect, aniridia, and developmental delay. Additional material on 11p13 was cytogenetically visible and array analyses identified a duplicated segment on 15q25‐26 chromosome region; further, array analyses revealed a small deletion (49 kb) at 11p13 region involving the ELP4 gene and a duplication at 8p23.1. Results were confirmed with both molecular and molecular cytogenetics techniques. Possibilities for etiological basis of clinical phenotype are discussed.

A Locus Identified on Chromosome18P11.31 is Associated with Hippocampal Abnormalities in a Family with Mesial Temporal Lobe Epilepsy

We aimed to identify the region harboring a putative candidate gene associated with hippocampal abnormalities (HAb) in a family with mesial temporal lobe epilepsy (MTLE). Genome-wide scan was performed in one large kindred with MTLE using a total of 332 microsatellite markers at ∼12 cM intervals. An additional 13 markers were genotyped in the candidate region. Phenotypic classes were defined according to the presence of hippocampal atrophy and/or hyperintense hippocampal T2 signal detected on magnetic resonance imaging. We identified a significant positive LOD score on chromosome 18p11.31 with a Zmax of 3.12 at D18S452. Multipoint LOD scores and haplotype analyses localized the candidate locus within a 6-cM interval flanked by D18S976 and D18S967. We present here evidence that HAb, which were previously related mainly to environmental risk factors, may be influenced by genetic predisposition. This finding may have major impact in the study of the mechanisms underlying abnormalities in mesial temporal lobe structures and their relationship with MTLE.

LINKGEN: a new algorithm to process data in genetic linkage studies.

Genetic linkage studies using whole genome scans are useful approaches for identifying genes related to human diseases. In general, these studies require genotyping of a large number of markers, which are used in statistical analysis. Recent technology has allowed easy genotyping of a large number of markers in less time; therefore, interface programs are required for manipulation of these large data sets. We present a new algorithm, which processes input data in LINKAGE format from data analyzed by automated genotyping systems. The algorithm was implemented in PERL script and R environment. Validation was performed with genotyped data from 127 individuals and 720 microsatellite markers of two whole genome scans. Our results showed a significant decrease in data processing time. In addition, this algorithm provides unbiased allele frequency estimation used for linkage analysis. LINKGEN is a freely available online tool and allows easier, faster, and reliable manipulation of large genotyping data sets.