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Malate is an important dicarboxylic acid produced from fumarate in the tricarboxylic acid cycle. Deficiencies of fumarate hydrolase (FH) and malate dehydrogenase (MDH), responsible for malate formation and metabolism, respectively, are known to cause recessive forms of neurodevelopmental disorders (NDDs). The malic enzyme isoforms, malic enzyme 1 (ME1) and 2 (ME2), are required for the conversion of malate to pyruvate. To date, there have been no reports linking deficiency of either malic enzyme isoforms to any Mendelian disease in humans. We report a patient presenting with NDD, subtle dysmorphic features, resolved dilated cardiomyopathy, and mild blood lactate elevation. Whole exome sequencing (WES) revealed a homozygous frameshift variant (c.1379_1380delTT, p.Phe460fs*22) in the malic enzyme 2 (ME2) gene resulting in truncated and unstable ME2 protein in vitro. Subsequent deletion of the yeast ortholog of human ME2 (hME2) resulted in growth arrest, which was rescued by overexpression of hME2, strongly supporting an important role of ME2 in mitochondrial function. Our results also support the pathogenicity and candidacy of the ME2 gene and variant in association with NDD. To our knowledge, this is the first report of a Mendelian human disease resulting from a biallelic variant in the ME encoding gene. Future studies are warranted to confirm ME2-associated recessive NDD., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful., (© The Author(s) 2023. Published by Oxford University Press.)

Background: The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear., Methods: Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n = 39) or with the wild-type N protein (RG patients n = 39) and compared to healthy controls (n = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells., Results: We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant., Conclusions: Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development., (© 2023. The Author(s).)

Purpose: Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified., Methods: Chromosomal analysis and exome sequencing were used to identify the genetic causes in 10 patients from 7 unrelated families with syndromic neurodevelopmental, seizure, and movement disorders and neurobehavioral phenotypes., Results: In total, 4 patients were found to have 3 different homozygous loss-of-function (LoF) variants, and 3 patients had 4 compound heterozygous missense variants in the candidate E3 ligase gene, HECTD4, that were rare, absent from controls as homozygous, and predicted to be deleterious in silico. In 3 patients from 2 families with Angelman-like syndrome, paralog-directed candidate gene approach detected 2 LoF variants in the other candidate E3 ligase gene, UBE3C, a paralog of the Angelman syndrome E3 ligase gene, UBE3A. The RNA studies in 4 patients with LoF variants in HECTD4 and UBE3C provided evidence for the LoF effect., Conclusion: HECTD4 and UBE3C are novel biallelic rare disease genes, expand the association of the other HECT E3 ligase group with neurodevelopmental syndromes, and could explain some of the missing heritability in patients with a suggestive clinical diagnosis of Angelman syndrome., Competing Interests: Conflict of Interest J.R.L. has stock ownership in 23andMe, is a paid consultant for Regeneron Genetics Center, and is a coinventor on multiple US and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders, and bacterial genomic fingerprinting. J.R.L. serves on the Scientific Advisory Board of Baylor Genetics. Baylor College of Medicine and Miraca Holdings have formed a joint venture with shared ownership and governance of Baylor Genetics, which performs clinical microarray analysis and other genomic studies (exome sequencing, genome sequencing) for patient and family care. N.A.M.A. is a paid consultant for Noor Diagnostics and Discovery, which performs genetic and genomic studies (exome sequencing, genome sequencing) for patient and family care. All other authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Objectives: Our objective was to identify the genetic cause in a family with a remarkable history of neurodevelopmental disease and growth retardation., Methods: A neurologic evaluation was performed, and DNA samples were obtained from the affected siblings and parents to perform whole-exome sequencing (WES)., Results: Both siblings presented with dysmorphic features, failure to thrive, global developmental delay, generalized hypotonia, feeding problems, and congenital heart disease. WES revealed a homozygous nonsense variant in the FRA10AC1 gene in both siblings., Discussion: A recent study has reported the first association of biallelic variants in the spliceosomal C complex gene, FRA10AC1 , with syndromic neurodevelopmental disease and growth retardation in 5 patients from 3 consanguineous families complex. In this study, we provide the first confirmation of the reported FRA10AC1 -related neurologic syndrome in an additional family., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Half of patients with a ciliopathy syndrome remain unsolved after initial analysis of whole exome sequencing (WES) data, highlighting the need for improved variant filtering and annotation. By candidate gene curation of WES data, combined with homozygosity mapping, we detected a homozygous predicted synonymous allele in NPHP3 in two children with hepatorenal fibrocystic disease from a consanguineous family. Analyses on patient-derived RNA shows activation of a cryptic mid-exon splice donor leading to frameshift. Remarkably, the same rare variant was detected in four additional families with hepatorenal disease from UK, US, and Saudi patient cohorts and in addition, another synonymous NPHP3 variant was identified in an unsolved case from the Genomics England 100,000 Genomes data set. We conclude that synonymous NPHP3 variants, not reported before and discarded by pathogenicity pipelines, solved several families with a ciliopathy syndrome. These findings prompt careful reassessment of synonymous variants, especially if they are rare and located in candidate genes., (© 2021 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Heterozygous pathogenic variants in SLC12A2 are reported in patients with nonsyndromic hearing loss. Recently, homozygous loss-of-function variants have been reported in two patients with syndromic intellectual disability, with or without hearing loss. However, the clinical and molecular spectrum of SLC12A2 disease has yet to be characterized and confirmed. Using whole-exome sequencing, we detected a homozygous splicing variant in four patients from two independent families with severe developmental delay, microcephaly, respiratory abnormalities, and subtle dysmorphic features, with or without congenital hearing loss. We also reviewed the reported cases with pathogenic variants associated with autosomal dominant and recessive forms of the SLC12A2 disease. About 50% of the cases have syndromic and nonsyndromic congenital hearing loss. All patients harboring the recessive forms of the disease presented with severe global developmental delay. Interestingly, all reported variants are located in the c-terminal domain, suggesting a critical role of this domain for the proper function of the encoded co-transporter protein. In conclusion, our study provides an additional confirmation of the autosomal recessive SLC12A2 disease.

Background: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner., Methods: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed nanopore sequencing of isothermal rapid viral amplification for near real-time analysis (NIRVANA). It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time., Findings: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per μL of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2-positive samples mirror the epidemiology of coronavirus disease 2019 (COVID-19). Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and pepper mild mottle virus (PMMoV) (an omnipresent virus and water-quality indicator) in municipal wastewater samples., Conclusions: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses., Funding: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01; M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436)., Competing Interests: A patent application based on methods described in this paper has been filed by King Abdullah University of Science and Technology, in which C.B. and M.L. are listed as inventors. The authors declare no other competing interests., (© 2021 Elsevier Inc.)

Background: Leukodystrophies (LDs) are inherited heterogeneous conditions that affect the central nervous system with or without peripheral nerve involvement. They are individually rare, but collectively, they are common. Thirty disorders were included by the Global Leukodystrophy Initiative Consortium (GLIA) as LDs. Methods: We conducted a retrospective chart review of a consecutive series of patients diagnosed with different types of LD from four large tertiary referral centers in Riyadh, Saudi Arabia. Only those 30 disorders defined by GLIA as LDs were included. Results: In total, 83 children from 61 families were identified and recruited for this study. The male-to-female ratio was 1.5:1, and a consanguinity rate of 58.5% was observed. An estimated prevalence of 1:48,780 or 2.05/100,000 was observed based on the clinical cohort, whereas a minimum of 1:32,857 or 3.04/100,000 was observed based on the local genetic database. The central region of the country exhibited the highest prevalence of LDs (48.5%). The most common LD was metachromatic leukodystrophy (MLD), and it accounted for 25.3%. The most common disorder based on carrier frequency was AGS. Novel variants were discovered in 51% of the cases, but 49% possessed previously reported variants. Missense variants were high in number and accounted for 73% of all cases. Compared with other disorders, MLD due to saposin b deficiency was more common than expected, Pelizaeus-Merzbacher-like disease was more prevalent than Pelizaeus-Merzbacher disease, and X-linked adrenoleukodystrophy was less common than expected. The mortality rate among our patients with LD was 24%. Conclusion: To the best of our knowledge, this is the largest cohort of patients with LD from Saudi Arabia. We present epidemiological, clinical, radiological, and genetic data. Furthermore, we report 18 variants that have not been reported previously. These findings are of great clinical and molecular utility for diagnosing and managing patients with LD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alfadhel, Almuqbil, Al Mutairi, Umair, Almannai, Alghamdi, Althiyab, Albarakati, Bashiri, Alshuaibi, Ba-Armah, Saleh, Al-Asmari, Faqeih, Altuwaijri, Al-Rumayyan, Balwi, Ababneh, Alswaid, Eyaid, Almontashiri, Alhashem, Hundallah, Bertoli-Avella, Bauer, Beetz, Alrifai, Alfares and Tabarki.)

Protein disulfide isomerase A6 (PDIA6) is an unfolded protein response (UPR)-regulating protein. PDIA6 regulates the UPR sensing proteins, Inositol requiring enzyme 1, and EIF2AK3. Biallelic inactivation of the two genes in mice and humans resulted in embryonic lethality, diabetes, skeletal defects, and renal insufficiency. We recently showed that PDIA6 inactivation in mice caused embryonic and early lethality, diabetes and immunodeficiency. Here, we present a case with asphyxiating thoracic dystrophy (ATD) syndrome and infantile-onset diabetes. Whole exome sequencing revealed a homozygous frameshift variant in the PDIA6 gene. RNA expression was reduced in a gene dosage-dependent manner, supporting a loss-of-function effect of this variant. Phenotypic correlation with the mouse model recapitulated the growth defect and delay, early lethality, coagulation, diabetes, immunological, and polycystic kidney disease phenotypes. In general, the phenotype of the current patient is consistent with phenotypes associated with the disruption of PDIA6 and the sensors of UPR in mice and humans. This is the first study to associate ATD to the UPR gene, PDIA6. We recommend screening ATD cases with or without insulin-dependent diabetes for variants in PDIA6., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To identify the genetic cause of a late-onset immunodeficiency and subacute progressive neurodegenerative disease affecting cognition, motor, visual, and cerebellar systems in a patient with a family history of 2 younger siblings with an early-onset immunodeficiency disease., Methods: Physical examinations, immunologic, brain MRI, whole-exome sequencing, and segregation studies were used to identify the genetic and neuroimmunologic etiology of disease in this family., Results: We identified a homozygous loss-of-function (LOF) mutation (c.271+1G>C) in the RFXANK gene in the index patient and one of his younger affected siblings. Biallelic mutations in the RFXANK gene are known to cause bare lymphocyte syndrome (BLS) type II, complementation group B. The clinical and immunologic investigations were consistent with a clinical diagnosis of BLS type II. MRI demonstrated global cerebral and cerebellar atrophy with white matter signal changes in the index case., Conclusions: In addition to BLS type II, our study has expanded and further characterized the phenotype associated with the LOF of RFXANK to include progressive neurodegenerative disease. Our study also provides evidence for the impact of LOF on brain development and function. Thus, early bone marrow transplantation, as a standard of care for BLS, could prove to be protective against the neurologic phenotypes in this group of patients., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Molecular diagnosis and surveillance of pathogens such as SARS-CoV-2 depend on nucleic acid isolation. Pandemics at the scale of COVID-19 can cause a global shortage of proprietary commercial reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open-source method, magnetic-nanoparticle-aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real-world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID-19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field-deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Global Challenges published by Wiley‐VCH GmbH.)

Biallelic variants in the USP53 gene have recently been reported to segregate with normal gamma glutamyltransferase (GGT) cholestasis. Using whole-exome sequencing (WES), we detected two USP53 homozygous variants (c.951delT; p. Phe317fs and c.1744C>T; p. Arg582*) in five additional cases, including an unpublished cousin of a previously described family with intractable itching and normal GGT cholestasis. Three patients, a child and two adults, presented with recurrent episodes of normal GGT cholestasis, consistent with a diagnosis of benign recurrent intrahepatic cholestasis (BRIC). Cholangiopathic changes, possibly autoimmune in origin, were recognized in some patients. Additional phenotypic details in one patient included an enlarged left kidney, and speech/developmental delay. Notably, two patients exhibited a complete response to rifampicin, and one responded to ursodeoxycholic acid (UDCA). Two adult patients were suspected to have autoimmune liver disease and treated with steroids. This report describes new cases of USP53 disease presenting with normal GGT cholestasis or BRIC in three children and two adults. We also describe the novel finding of a dramatic response to rifampicin. The association of cholangiopathy with normal GGT cholestasis provides a diagnostic challenge and remains poorly understood.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread globally. Although several rapid commercial serological assays have been developed, little is known about their performance and accuracy in detecting SARS-CoV-2-specific antibodies in COVID-19 patient samples. Here, we have evaluated the performance of seven commercially available rapid lateral flow immunoassays (LFIA) obtained from different manufacturers, and compared them to in-house developed and validated ELISA assays for the detection of SARS-CoV-2-specific IgM and IgG antibodies in RT-PCR-confirmed COVID-19 patients. While all evaluated LFIA assays showed high specificity, our data showed a significant variation in sensitivity of these assays, which ranged from 0% to 54% for samples collected early during infection (3-7 days post symptoms onset) and from 54% to 88% for samples collected at later time points during infection (8-27 days post symptoms onset). Therefore, we recommend prior evaluation and validation of these assays before being routinely used to detect IgM and IgG in COVID-19 patients. Moreover, our findings suggest the use of LFIA assays in combination with other standard methods, and not as an alternative.

The Coronavirus Disease 2019 (COVID-19), caused by SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Antigen-specific responses are of unquestionable value for clinical management of COVID-19 patients. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized COVID-19 patients with different disease presentations (i.e., mild, moderate or severe), need for intensive care units (ICU) admission or outcomes (i.e., survival vs death). We show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Interestingly, significantly higher levels of nAbs as well as anti-S1 and -N IgG and IgM antibodies were found in patients with more severe symptoms, patients requiring admission to ICU or those with fatal outcomes. More importantly, early after symptoms onset, we found that the levels of anti-N antibodies correlated strongly with disease severity. Collectively, these findings provide new insights into the kinetics of antibody responses in COVID-19 patients with different disease severity.

Purpose: Asparagine synthetase deficiency (ASNSD) is a rare neurometabolic disease. Patients may not demonstrate low asparagine levels, which highlights the advantage of molecular over biochemical testing in the initial work-up of ASNSD. We aimed to further delineate the ASNSD variant and phenotypic spectrum and determine the value of biochemical testing as a frontline investigation in ASNSD., Methods: We retrospectively collected the clinical and molecular information on 13 families with ASNSD from the major metabolic clinics in Saudi Arabia., Results: The major phenotypes included congenital microcephaly (100%), facial dysmorphism (100%), global developmental delay (100%), brain abnormalities (100%), spasticity (86%), and infantile-onset seizures (93%). Additional unreported phenotypes included umbilical hernia, osteopenia, eczema, lung hypoplasia, and hearing loss. Overall, seven homozygous variants accounted for ASNSD. The p.Tyr398Cys and p.Asn75Ile variants accounted for 54% of the cases. The clinical sensitivity and specificity of the proposed biochemical analysis of cerebrospinal fluid (CSF) for the detection of patients with ASNSD were 83% and 98%, respectively., Conclusion: Our study describes the largest reported ASNSD cohort with clinical, molecular, and biochemical characterization. Taking into consideration the suboptimal sensitivity of biochemical screening, the delineation of the phenotype variant spectrum is of diagnostic utility for accurate diagnosis, prognosis, counseling, and carrier screening.

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

Global untargeted metabolomics (GUM) has entered clinical diagnostics for genetic disorders. We compared the clinical utility of GUM with traditional targeted metabolomics (TM) as a screening tool in patients with established genetic disorders and determined the scope of GUM as a discovery tool in patients with no diagnosis under investigation. We compared TM and GUM data in 226 patients. The first cohort (n = 87) included patients with confirmed inborn errors of metabolism (IEM) and genetic syndromes; the second cohort (n = 139) included patients without diagnosis who were undergoing evaluation for a genetic disorder. In patients with known disorders (n = 87), GUM performed with a sensitivity of 86% (95% CI: 78-91) compared with TM for the detection of 51 diagnostic metabolites. The diagnostic yield of GUM in patients under evaluation with no established diagnosis (n = 139) was 0.7%. GUM successfully detected the majority of diagnostic compounds associated with known IEMs. The diagnostic yield of both targeted and untargeted metabolomics studies is low when assessing patients with non-specific, neurological phenotypes. GUM shows promise as a validation tool for variants of unknown significance in candidate genes in patients with non-specific phenotypes.

Retinal arterial macroaneurysms with supravalvular pulmonic stenosis (RAMSVPS), also known as Familial Retinal Arterial Macroaneurysms (FRAM) syndrome, is a very rare multisystem disorder. Here, we present a case series comprising ophthalmologic and systemic evaluation of patients homozygous for RAMSVPS syndrome causative IGFBP7 variant. New clinical details on 22 previously published and 8 previously unpublished patients are described. Age at first presentation ranged from 1 to 34 years. The classical feature of macroaneurysms and vascular beading involving the retinal arteries was universal. Follow up extending up to 14 years after initial diagnosis revealed recurrent episodes of bleeding and leakage from macroaneurysms in 55% and 59% of patients, respectively. The majority of patients who underwent echocardiography (18/23) showed evidence of heart involvement, most characteristically pulmonary (valvular or supravalvular) stenosis, often requiring surgical correction (12/18). Four patients died in the course of the study from complications of pulmonary stenosis, cerebral hemorrhage, and cardiac complications. Liver involvement (usually cirrhosis) was observed in eight patients. Cerebral vascular involvement was observed in one patient, and stroke was observed in two. We conclude that RAMSVPS is a recognizable syndrome characterized by a high burden of ocular and systemic morbidity, and risk of premature death. Recommendations are proposed for early detection and management of these complications., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

PurposeHearing loss is more prevalent in the Saudi Arabian population than in other populations; however, the full range of genetic etiologies in this population is unknown. We report the genetic findings from 33 Saudi hearing-loss probands of tribal ancestry, with predominantly prelingual severe to profound hearing loss.MethodsTesting was performed over the course of 2012-2016, and involved initial GJB2 sequence and GJB6-D13S1830 deletion screening, with negative cases being reflexed to a next-generation sequencing panel with 70, 71, or 87 hearing-loss genes.ResultsA "positive" result was reached in 63% of probands, with two recurrent OTOF variants (p.Glu57* and p.Arg1792His) accountable for a third of all "positive" cases. The next most common cause was pathogenic variants in MYO7A and SLC26A4, each responsible for three "positive" cases. Interestingly, only one "positive" diagnosis had a DFNB1-related cause, due to a homozygous GJB6-D13S1830 deletion, and no sequence variants in GJB2 were detected.ConclusionOur findings implicate OTOF as a potential major contributor to hearing loss in the Saudi population, while highlighting the low contribution of GJB2, thus offering important considerations for clinical testing strategies for Saudi patients. Further screening of Saudi patients is needed to characterize the genetic spectrum in this population.

The 9p21.3 risk locus is the first locus to be associated with an increased risk of coronary artery disease (CAD)-related events and many other phenotypes. This locus contains 59 single nucleotide polymorphisms (SNPs) in a region with multiple long range enhancers and long non-coding RNAs (lncRNAs) that affect the expression of neighbouring genes, cyclin-dependent kinase 2A and 2B (CDKN2A and CDKN2B), which are required for controlling vascular smooth muscle cell proliferation and ageing. Several studies have attempted to identify the precise mechanism by which this locus exerts its pathogenic effect to increase the risk of CAD-related events. In this review, we will highlight the major advances in our understanding of the genotype-phenotype correlation at the mechanistic and phenotypic levels. The high population attributable risk of the 9p21.3 risk locus, mechanistic knowledge acquired thus far, and ongoing research efforts could facilitate the design of novel therapeutic molecules to reduce the risk of CAD and its related events.