Copy number variations in a Brazilian cohort with autism spectrum disorders highlight the contribution of cell adhesion genes.

Prediction of pathogenicity of rare copy number variations (CNVs), a genomic alteration known to contribute to the etiology of autism spectrum disorder (ASD), represents a serious limitation to interpreting genetic tests, particularly for genetic counseling purposes. Chromosomal microarray analysis (CMA) was conducted in a unique collection of 144 Brazilian individuals with ASD of strong European and African ancestries. Rare CNVs were detected in 39 patients: 41 of unknown significance (VUS), four pathogenic and one likely pathogenic CNVs (clinical yield of 4.1%; 5/122). Based on gene content and recurrence in three large cohorts [a Brazilian neurodevelopmental disorder cohort, the autism MSSNG cohort, and the Canadian-based Centre for Applied Genomics microarray database], this work strengthened the pathogenicity of 14 genes (FAT1, CAMK4, BIRC6, DPP6, CSMD1, CTNNA3, CDH8/CDH11, CDH13, OR1C1, CNTN6, CNTNAP4, FGF2 and PTPRN2) within 14 CNVs. Notably, enrichment of cell adhesion proteins to ASD etiology was identified (p < 0.05), highlighting the importance of these gene families in the etiology of ASD.

Newborn Screening for 5q Spinal Muscular Atrophy: Comparisons between Real-Time PCR Methodologies and Cost Estimations for Future Implementation Programs.

Since the approval of modifying therapies for Spinal Muscular Atrophy (SMA), several protocols aiming to screen SMN1 homozygous deletion in a neonatal context have been published. However, no work has compared different methodologies along with detailed implementation costs for centers where the neonatal screening of SMA has not yet been implemented. Therefore, our work compared different qualitative real-time PCR approaches for SMA screening and the estimated costs of test implementation. Using Brazilian blood samples, the presence and absence (P/A) and melt curve protocols were analyzed. MLPA was used as a confirmatory test. The costs were calculated for the simplex and multiplex tests plus equipment. The test workflow was based on the present experience and literature report. The accuracy of the P/A protocol was 1 (95% CI 0.8677-1) using dried blood spots (DBS). The melt curve protocol also achieved 100% concordance. The consumable costs ranged from USD 1.68 to 4.42 and from USD 2.04 to 12.76 per reaction, for the simplex and multiplex tests, respectively. The equipment acquisition costs ranged from USD 44,817.07 to 467,253.10, with several factors influencing this value presented. Our work presents a framework for decision-making, with a project demonstration of the different assays that will be useful in dealing with the issues of cost and availability of reagents. Moreover, we present a literature review and discussion of important concerns regarding treatment policies. We take the first step towards a future SMA NBS pilot program where it is not yet a reality.

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread.

Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >102 copies/µL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.

Neuroprogenitor Cells From Patients With TBCK Encephalopathy Suggest Deregulation of Early Secretory Vesicle Transport.

Biallelic pathogenic variants in TBCK cause encephaloneuropathy, infantile hypotonia with psychomotor retardation, and characteristic facies 3 (IHPRF3). The molecular mechanisms underlying its neuronal phenotype are largely unexplored. In this study, we reported two sisters, who harbored biallelic variants in TBCK and met diagnostic criteria for IHPRF3. We provided evidence that TBCK may play an important role in the early secretory pathway in neuroprogenitor cells (iNPC) differentiated from induced pluripotent stem cells (iPSC). Lack of functional TBCK protein in iNPC is associated with impaired endoplasmic reticulum-to-Golgi vesicle transport and autophagosome biogenesis, as well as altered cell cycle progression and severe impairment in the capacity of migration. Alteration in these processes, which are crucial for neurogenesis, neuronal migration, and cytoarchitecture organization, may represent an important causative mechanism of both neurodevelopmental and neurodegenerative phenotypes observed in IHPRF3. Whether reduced mechanistic target of rapamycin (mTOR) signaling is secondary to impaired TBCK function over other secretory transport regulators still needs further investigation.

Waardenburg syndrome: Novel mutations in a large Brazilian sample.

This paper deals with the molecular investigation of Waardenburg syndrome (WS) in a sample of 49 clinically diagnosed probands (most from southeastern Brazil), 24 of them having the type 1 (WS1) variant (10 familial and 14 isolated cases) and 25 being affected by the type 2 (WS2) variant (five familial and 20 isolated cases). Sequential Sanger sequencing of all coding exons of PAX3, MITF, EDN3, EDNRB, SOX10 and SNAI2 genes, followed by CNV detection by MLPA of PAX3, MITF and SOX10 genes in selected cases revealed many novel pathogenic variants. Molecular screening, performed in all patients, revealed 19 causative variants (19/49 = 38.8%), six of them being large whole-exon deletions detected by MLPA, seven (four missense and three nonsense substitutions) resulting from single nucleotide substitutions (SNV), and six representing small indels. A pair of dizygotic affected female twins presented the c.430delC variant in SOX10, but the mutation, imputed to gonadal mosaicism, was not found in their unaffected parents. At least 10 novel causative mutations, described in this paper, were found in this Brazilian sample. Copy-number-variation detected by MLPA identified the causative mutation in 12.2% of our cases, corresponding to 31.6% of all causative mutations. In the majority of cases, the deletions were sporadic, since they were not present in the parents of isolated cases. Our results, as a whole, reinforce the fact that the screening of copy-number-variants by MLPA is a powerful tool to identify the molecular cause in WS patients.

Dystrophin Is Required for Proper Functioning of Luminance and Red-Green Cone Opponent Mechanisms in the Human Retina.

PURPOSE: Visual information is processed in parallel pathways in the visual system. Parallel processing begins at the synapse between the photoreceptors and their postreceptoral neurons in the human retina. The integrity of this first neural connection is vital for normal visual processing downstream. Of the numerous elements necessary for proper functioning of this synaptic contact, dystrophin proteins in the eye play an important role. Deficiency of muscle dystrophin causes Duchenne muscular dystrophy (DMD), an X-linked disease that affects muscle function and leads to decreased life expectancy. In DMD patients, postreceptoral retinal mechanisms underlying scotopic and photopic vision and ON- and OFF-pathway responses are also altered. METHODS: In this study, we recorded the electroretinogram (ERG) while preferentially activating the (red-green) opponent or the luminance pathway, and compared data from healthy participants (n = 16) with those of DMD patients (n = 10). The stimuli were heterochromatic sinusoidal modulations at a mean luminance of 200 cd/m2. The recordings allowed us also to analyze ON and OFF cone-driven retinal responses. RESULTS: We found significant differences in 12-Hz response amplitudes and phases between controls and DMD patients, with conditions with large luminance content resulting in larger response amplitudes in DMD patients compared to controls, whereas responses of DMD patients were smaller when pure chromatic modulation was given. CONCLUSIONS: The results suggest that dystrophin is required for the proper function of luminance and red-green cone opponent mechanisms in the human retina.

Use of PCR reactions (polymerase chain reaction) on Trichomonas vaginalis diagnosis in patients from Araraquara region (State of Säo Paulo, Brazil)

Trichomonas vaginalis é um protozoário flagelado da família TRICHOMONADIDAE, responsável por uma doença que ataca o sistema genito-urinário, causando, em mulheres, vaginites e cervicites, dentre outras complicaçöes, e, em homens, prostatites, uretrites e síndrome genito-urinárias menores. Em funçäo de que diagnósticos precisos säo uma importante ferramenta para o tratamento apropriado e prevençäo da transmissäo da doença,este trabalho possibilitou a padronizaçäo da reaçäo de PCR e avaliaçäo de seu emprego no diagnóstico deste parasita. Na padronizaçäo da reaçäo, utilizou-se DNA genômico de diferentes cepas de T. vaginalis mantidas em laboratório e o protocolo da PCR foi realizado de acordo com os parâmetros propostos por Riley e colaboradores. Secreçöes vaginais foram coletadas e amplificadas pela mesma metodologia. Cento e oitenta amostras clínicas foram analisadas por 3 métodos diferentes, o PCR e os métodos tradicionais exame a fresco e cultura do parasita em meio de Diamond. Dessas amostras, 6 foram positivas pela PCR, das quais duas foram detectadas apenas por PCR, uma por PCR e cultura, duas por PCR e exame fresco e uma pelos 3 métodos usados. Esses resultados demonstraram que a reaçäo de PCR pode ser convenientemente padronizada nas condiçöes do laboratório e seu uso efetivo como método diagnóstico de tricomoníase foi confirmado.