Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis.

Vesicle-associated membrane protein-associated protein-B (VAPB) is an ER membrane bound protein. VAPB P56S causes a dominant, familial form of amyotrophic lateral sclerosis (ALS), however, the mechanism through which this mutation causes motor neuron (MN) disease remains unknown. Using inducible wild type (WT) and VAPB P56S expressing iPSC-derived MNs we show that VAPB P56S, but not WT, protein decreased neuronal firing and mitochondrial-ER contact (MERC) with an associated age-dependent decrease in mitochondrial membrane potential (MMP); all typical characteristics of MN-disease. We further show that VAPB P56S expressing iPSC-derived MNs have enhanced age-dependent sensitivity to ER stress. We identified elevated expression of the master regulator of the Integrated Stress Response (ISR) marker ATF4 and decreased protein synthesis in the VAPB P56S iPSC-derived MNs. Chemical inhibition of ISR with the compound, ISRIB, rescued all MN disease phenotype in VAPB P56S MNs. Thus, our results not only support ISR inhibition as a potential therapeutic target for ALS patients, but also provides evidence to pathogenesis.

Copy number variation in pituitary stalk interruption syndrome: A large case series of sporadic non-syndromic patients and literature review.

Abnormal hypothalamic/posterior pituitary development appears to be a major determinant of pituitary stalk interruption syndrome (PSIS). The observation of familial cases and associated congenital abnormalities suggests a genetic basis. Single-gene mutations explain less than 5% of the cases, and whole exome sequencing has shown heterogeneous results. The present study aimed to assess copy number variation (CNV) using array-based comparative genomic hybridization (aCGH) in patients with non-syndromic PSIS and comprehensively review data from the literature on CNV analysis in congenital hypopituitarism (CH) patients. Twenty-one patients with sporadic CH from our outpatient clinics presented with ectopic posterior pituitary (EPP) and no central nervous system abnormalities on magnetic resonance image (MRI) or any other malformations on physical examination at presentation were enrolled in the study. aCGH using a whole-genome customized 400K oligonucleotide platform was performed in our patients. For the literature review, we searched for case reports of patients with CH and CNV detected by either karyotype or aCGH reported in PubMed up to November 2021. Thirty-five distinct rare CNVs were observed in 18 patients (86%) and two of them (6%) were classified as pathogenic: one deletion of 1.8 Mb in chromosome 17 (17q12) and one deletion of 15 Mb in chromosome 18 (18p11.32p11.21), each one in a distinct patient. In the literature review, 67 pathogenic CNVs were published in 83 patients with CH, including the present study. Most of these patients had EPP (78% out of the 45 evaluated by sellar MRI) and were syndromic (70%). The most frequently affected chromosomes were X, 18, 20 and 1. Our study has found that CNV can be a mechanism of genetic abnormality in non-syndromic patients with CH and EPP. In future studies, one or more genes in those CNVs, both pathogenic and variant of uncertain significance, may be considered as good candidate genes.

Small-fibre Neuropathy in Patients with Familial Amyotrophic Lateral Sclerosis Type 8.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a degenerative disease of the nervous system that primarily affects motor neurons. ALS type 8 (ALS8) is a familiar form with predominant involvement of lower motor neurons, tremor, and slow progression. OBJECTIVE: The aim of this study was to describe sensory involvement in a cohort of ALS8 patients and compare it with the characteristics of sporadic ALS (sALS) patients and controls. METHODS: We compared data from 40 ALS8 and 10 sALS patients assessed by neurological evaluation and electrophysiological study. Skin biopsies were performed in these patients and 12 controls for analysis of intraepidermal nerve fiber (IENF) density by protein gene product 9.5 (PGP 9.5) immunohistochemistry. RESULTS: The ALS8 group was younger than the sALS group at the onset of symptoms (p < 0.05) and had a longer disease evolution (p < 0.01). Sensory abnormalities were evident in 35% of the ALS8 and 30% of the sALS patients by neurological examination, and all ALS patients presented normal sensory nerve action potentials. Despite being similar in the ALS8 and sALS groups, IENF density in the ALS8 group was lower than that in the controls (p < 0.0005). In the ALS8 group, IENF density was significantly lower in patients with impairment of vibratory sensation than in those without this finding (p < 0.05) and in females than in males (p < 0.05). CONCLUSION: Sensory impairment and decreased IENF density are present in ALS8 patients at a frequency and intensity similar to that in the sALS group.

Genetic risk factors and COVID-19 severity in Brazil: results from BRACOVID study.

The coronavirus disease 2019 (COVID-19) pandemic has changed the paradigms for disease surveillance and rapid deployment of scientific-based evidence for understanding disease biology, susceptibility and treatment. We have organized a large-scale genome-wide association study (GWAS) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected individuals in Sao Paulo, Brazil, one of the most affected areas of the pandemic in the country, itself one of the most affected in the world. Here, we present the results of the initial analysis in the first 5233 participants of the BRACOVID study. We have conducted a GWAS for COVID-19 hospitalization enrolling 3533 cases (hospitalized COVID-19 participants) and 1700 controls (non-hospitalized COVID-19 participants). Models were adjusted by age, sex and the 4 first principal components. A meta-analysis was also conducted merging BRACOVID hospitalization data with the Human Genetic Initiative (HGI) Consortia results. BRACOVID results validated most loci previously identified in the HGI meta-analysis. In addition, no significant heterogeneity according to ancestral group within the Brazilian population was observed for the two most important COVID-19 severity associated loci: 3p21.31 and Chr21 near IFNAR2. Using only data provided by BRACOVID, a new genome-wide significant locus was identified on Chr1 near the genes DSTYK and RBBP5. The associated haplotype has also been previously associated with a number of blood cell related traits and might play a role in modulating the immune response in COVID-19 cases.

MHC Variants Associated With Symptomatic Versus Asymptomatic SARS-CoV-2 Infection in Highly Exposed Individuals.

Despite the high number of individuals infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) symptoms worldwide, many exposed individuals remain asymptomatic and/or uninfected and seronegative. This could be explained by a combination of environmental (exposure), immunological (previous infection), epigenetic, and genetic factors. Aiming to identify genetic factors involved in immune response in symptomatic COVID-19 as compared to asymptomatic exposed individuals, we analyzed 83 Brazilian couples where one individual was infected and symptomatic while the partner remained asymptomatic and serum-negative for at least 6 months despite sharing the same bedroom during the infection. We refer to these as "discordant couples". We performed whole-exome sequencing followed by a state-of-the-art method to call genotypes and haplotypes across the highly polymorphic major histocompatibility complex (MHC) region. The discordant partners had comparable ages and genetic ancestry, but women were overrepresented (65%) in the asymptomatic group. In the antigen-presentation pathway, we observed an association between HLA-DRB1 alleles encoding Lys at residue 71 (mostly DRB1*03:01 and DRB1*04:01) and DOB*01:02 with symptomatic infections and HLA-A alleles encoding 144Q/151R with asymptomatic seronegative women. Among the genes related to immune modulation, we detected variants in MICA and MICB associated with symptomatic infections. These variants are related to higher expression of soluble MICA and low expression of MICB. Thus, quantitative differences in these molecules that modulate natural killer (NK) activity could contribute to susceptibility to COVID-19 by downregulating NK cell cytotoxic activity in infected individuals but not in the asymptomatic partners.

Exome sequencing and targeted gene panels: a simulated comparison of diagnostic yield using data from 158 patients with rare diseases.

Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.

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.

Frequency of carriers for rare recessive Mendelian diseases in a Brazilian cohort of 320 patients.

Several Mendelian disorders follow an autosomal recessive inheritance pattern. Epidemiological information on many inherited disorders may be useful to guide health policies for rare diseases, but it is often inadequate, particularly in developing countries. We aimed to calculate the carrier frequencies of rare autosomal recessive Mendelian diseases in a cohort of Brazilian patients using whole exome sequencing (WES). We reviewed the molecular findings of WES from 320 symptomatic patients who had carrier status for recessive diseases. Using the Hardy-Weinberg equation, we estimated recessive disease frequencies (q2 ) considering the respective carrier frequencies (2pq) observed in our study. We calculated the sensitivity of carrier screening tests based on lists of genes from five different clinical laboratories that offer them in Brazil. A total of 425 occurrences of 351 rare variants were reported in 278 different genes from 230 patients (71.9%). Almost half (48.8%) were carriers of at least one heterozygous pathogenic/likely pathogenic variant for rare metabolic disorders, while 25.9% of epilepsy, 18.1% of intellectual disabilities, 15.6% of skeletal disorders, 10.9% immune disorders, and 9.1% of hearing loss. We estimated that an average of 67% of the variants would not have been detected by carrier screening panels. The combined frequencies of autosomal recessive diseases were estimated to be 26.39/10,000 (or ~0.26%). This study shows the potential research utility of WES to determine carrier status, which may be a possible strategy to evaluate the clinical and social burden of recessive diseases at the population level and guide the optimization of carrier screening panels.

Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.

Different gene expression profiles in iPSC-derived motor neurons from ALS8 patients with variable clinical courses suggest mitigating pathways for neurodegeneration.

Amyotrophic lateral sclerosis type 8 (ALS8) is an autosomal dominant form of ALS, which is caused by pathogenic variants in the VAPB gene. Here we investigated five ALS8 patients, classified as 'severe' and 'mild' from a gigantic Brazilian kindred, carrying the same VAPB mutation but displaying different clinical courses. Copy number variation and whole exome sequencing analyses in such individuals ruled out previously described genetic modifiers of pathogenicity. After deriving induced pluripotent stem cells (iPSCs) for each patient (N = 5) and controls (N = 3), motor neurons were differentiated, and high-throughput RNA-Seq gene expression measurements were performed. Functional cell death and oxidative metabolism assays were also carried out in patients' iPSC-derived motor neurons. The degree of cell death and mitochondrial oxidative metabolism were similar in iPSC-derived motor neurons from mild patients and controls and were distinct from those of severe patients. Similar findings were obtained when RNA-Seq from such cells was performed. Overall, 43 genes were upregulated and 66 downregulated in the two mild ALS8 patients when compared with severe ALS8 individuals and controls. Interestingly, significantly enriched pathways found among differentially expressed genes, such as protein translation and protein targeting to the endoplasmic reticulum (ER), are known to be associated with neurodegenerative processes. Taken together, the mitigating mechanisms here presented appear to maintain motor neuron survival by keeping translational activity and protein targeting to the ER in such cells. As ALS8 physiopathology has been associated with proteostasis mechanisms in ER-mitochondria contact sites, such differentially expressed genes appear to relate to the bypass of VAPB deficiency.

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases.

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

A novel GNRHR gene mutation causing congenital hypogonadotrophic hypogonadism in a Brazilian kindred.

Congenital hypogonadotrophic hypogonadism (CHH) is a challenging inherited endocrine disorder characterised by absent or incomplete pubertal development and infertility as a result of the low action/secretion of the hypothalamic gonadotrophin-releasing hormone (GnRH). Given a growing list of gene mutations accounting for CHH, the application of massively parallel sequencing comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. The present study proposes the use of whole exome sequencing (WES) to identify causative and modifying mutations based on a phenotype-genotype CHH analysis using an in-house exome pipeline. Based on 44 known genes related to CHH in humans, we were able to identify a novel homozygous gonadotrophin-releasing hormone receptor (GNRHR) p.Thr269Met mutant, which segregates with the CHH kindred and was predicted to be deleterious by in silico analysis. A functional study measuring intracellular inositol phosphate (IP) when stimulated with GnRH on COS-7 cells confirmed that the p.Thr269Met GnRHR mutant performed greatly diminished IP accumulation relative to the transfected wild-type GnRHR. Additionally, the proband carries three heterozygous variants in CCDC141 and one homozygous in SEMA3A gene, although their effects with respect to modifying the phenotype are uncertain. Because they do not segregate with reproductive phenotype in family members, we advocate they do not contribute to CHH oligogenicity. WES proved to be useful for CHH molecular diagnosis and reinforced its benefit with respect to identifying heterogeneous genetic disorders. Our findings expand the GnRHR mutation spectrum and phenotype-genotype correlation in CHH.

Mutational profiling of acute myeloid leukemia with normal cytogenetics in Brazilian patients: the value of next-generation sequencing for genomic classification.

Karyotype (KT) aberrations are important prognostic factors for acute myeloid leukemia (AML); however, around 50% of cases present normal results. Single nucleotide polymorphism array can detect chromosomal gains, losses or uniparental disomy that are invisible to KT, thus improving patients' risk assessment. However, when both tests are normal, important driver mutations can be detected by the use of next-generation sequencing (NGS). Fourteen adult patients with AML with normal cytogenetics were investigated by NGS for 19 AML-related genes. Every patient presented at least one mutation: DNMT3A in nine patients; IDH2 in six; IDH1 in three; NRAS and NPM1 in two; and TET2, ASXL1, PTPN11, and RUNX1 in one patient. No mutations were found in FLT3, KIT, JAK2, CEBPA, GATA2, TP53, BRAF, CBL, KRAS, and WT1 genes. Twelve patients (86%) had at least one mutation in genes related with DNA methylation (DNMT3A, IDH1, IDH2, and TET2), which is involved in regulation of gene expression and genomic stability. All patients could be reclassified based on genomic status and nine had their prognosis modified. In summary, NGS offers insights into the molecular pathogenesis and biology of cytogenetically normal AML in Brazilian patients, indicating that the prognosis could be further stratified by different mutation combinations. This study shows a different frequency of mutations in Brazilian population that should be confirmed.

DUOX2 Mutations Are Associated With Congenital Hypothyroidism With Ectopic Thyroid Gland.

Context: Thyroid dysgenesis (TD) is the leading cause of congenital hypothyroidism (CH). The etiology of TD remains unknown in ∼90% of cases, the most common form being thyroid ectopia (TE) (48% to 61%). Objective: To search for candidate genes in hypothyroid children with TE. Design, Setting, and Participants: We followed a cohort of 268 children with TD and performed whole-exome sequencing (WES) in three children with CH with TE (CHTE) and compared them with 18 thyroid-healthy controls. We then screened an additional 41 children with CHTE by Sanger sequencing and correlated the WES and Sanger molecular findings with in vitro functional analysis. Main Outcome Measures: Genotyping, mutation prediction analysis, and in vitro functional analysis. Results: We identified seven variants in the DUOX2 gene, namely G201E, L264CfsX57, P609S, M650T, E810X, M822V, and E1017G, and eight known variations. All children carrying DUOX2 variations had high thyroid-stimulating hormone levels at neonatal diagnosis. All mutations were localized in the N-terminal segment, and three of them led to effects on cell surface targeting and reactive oxygen species generation. The DUOX2 mutants also altered the interaction with the maturation factor DUOXA2 and the formation of a stable DUOX2/DUOXA2 complex at the cell surface, thereby impairing functional enzymatic activity. We observed no mutations in the classic genes related to TD or in the DUOX1 gene. Conclusion: Our findings suggest that, in addition to thyroid hormonogenesis, the DUOX2 N-terminal domain may play a role in thyroid development.

Development and validation of a variant detection workflow for BRCA1 and BRCA2 genes and its clinical application based on the Ion Torrent technology.

BACKGROUND: Breast cancer is the most common among women worldwide, and ovarian cancer is the most difficult gynecological tumor to diagnose and with the lowest chance of cure. Mutations in BRCA1 and BRCA2 genes increase the risk of ovarian cancer by 60% and breast cancer by up to 80% in women. Molecular tests allow a better orientation for patients carrying these mutations, affecting prophylaxis, treatment, and genetic counseling. RESULTS: Here, we evaluated the performance of a panel for BRCA1 and BRCA2, using the Ion Torrent PGM (Life Technologies) platform in a customized workflow and multiplex ligation-dependent probe amplification for detection of mutations, insertions, and deletions in these genes. We validated the panel with 26 samples previously analyzed by Myriad Genetics Laboratory, and our workflow showed 95.6% sensitivity and 100% agreement with Myriad reports, with 85% sensitivity on the positive control sample from NIST. We also screened 68 clinical samples and found 22 distinct mutations. CONCLUSIONS: The selection of a robust methodology for sample preparation and sequencing, together with bioinformatics tools optimized for the data analysis, enabled the development of a very sensitive test with high reproducibility. We also highlight the need to explore the limitations of the NGS technique and the strategies to overcome them in a clinically confident manner.

Different Donors Mesenchymal Stromal Cells Secretomes Reveal Heterogeneous Profile of Relevance for Therapeutic Use.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by null mutations in the dystrophin gene. Although the primary defect is the deficiency of muscle dystrophin, secondary events, including chronic inflammation, fibrosis, and muscle regeneration failure are thought to actively contribute to disease progression. Despite several advances, there is still no effective therapy for DMD. Therefore, the potential regenerative capacities, and immune-privileged properties of mesenchymal stromal cells (MSCs), have been the focus of intense investigation in different animal models aiming the treatment of these disorders. However, these studies have shown different outcomes according to the sources from which MSCs were obtained, which raise the question whether stem cells from distinct sources have comparable clinical effects. Here, we analyzed the protein content of the secretome of MSCs, isolated from three different sources (adipose tissue, skeletal muscle, and uterine tubes), obtained from five donors and evaluated their in vitro properties when cocultured with DMD myoblasts. All MSC lineages showed pathways enrichment related to protein metabolic process, oxidation-reduction process, cell proliferation, and regulation of apoptosis. We found that MSCs secretome proteins and their effect in vitro vary significantly according to the tissue and donors, including opposite effects in apoptosis assay, indicating the importance of characterizing MSC secretome profile before its use in animal and clinical trials. Despite the individual differences a pool of conditioned media from all MSCs lineages was able to delay apoptosis and enhance migration when in contact with DMD myoblasts.