Identification of a DNA methylation signature for Renpenning syndrome (RENS1), a spliceopathy.

The challenges and ambiguities in providing an accurate diagnosis for patients with neurodevelopmental disorders have led researchers to apply epigenetics as a technique to validate the diagnosis provided based on the clinical examination and genetic testing results. Genome-wide DNA methylation analysis has recently been adapted for clinical testing of patients with genetic neurodevelopmental disorders. In this paper, preliminary data demonstrating a DNA methylation signature for Renpenning syndrome (RENS1 - OMIM 309500), which is an X-linked recessive neurodevelopmental disorder caused by variants in polyglutamine-binding protein 1 (PQBP1) is reported. The identified episignature was then utilized to construct a highly sensitive and specific binary classification model. Besides providing evidence for the existence of a DNA methylation episignature for Renpenning syndrome, this study increases the knowledge of the molecular mechanisms related to the disease. Moreover, the availability of more subjects in future may facilitate the establishment of an episignature that can be utilized for diagnosis in a clinical setting and for reclassification of variants of unknown clinical significance.

Feasibility of Screening for Chromosome 15 Imprinting Disorders in 16 579 Newborns by Using a Novel Genomic Workflow.

Importance: Newborn screening for Angelman syndrome (AS), Prader-Willi syndrome (PWS), and chromosome 15 duplication syndrome (Dup15q) may lead to benefit from early diagnosis and treatment. Objective: To examine the feasibility of newborn screening for these chromosome 15 imprinting disorders at population scale. Design, Setting, and Participants: In this diagnostic study, the validation data set for the first-tier SNRPN test, called methylation-specific quantitative melt analysis (MS-QMA), included 109 PWS, 48 AS, 9 Dup15q, and 1190 population control newborn blood spots (NBS) and peripheral tissue samples from participants recruited from January 2000 to December 2016. The test data set included NBS samples from 16 579 infants born in 2011. Infants with an NBS identified as positive for PWS, AS, or Dup15q by the first-tier test were referred for droplet digital polymerase chain reaction, real-time polymerase chain reaction, and low-coverage whole-genome sequencing for confirmatory testing. Data analyses were conducted between February 12, 2015, and August 15, 2020. Results: In the validation data set, the median age for the 77 patients with PWS was 3.00 years (IQR, 0.01-44.50 years); for the 46 patients with AS, 2.76 years (IQR, 0.028 to 49.00 years); and for the 9 patients with Dup15q, 4.00 years (IQR, 1.00 to 28.00 years). Thirty-eight patients (51.4%) in the PWS group, 20 patients (45.5%) in the AS group, and 6 patients (66.7%) in the Dup15q group who had sex reported were male. The validation data set showed MS-QMA sensitivity of 99.0% for PWS, 93.8% for AS, and 77.8% for Dup15q; specificity of 100% for PWS, AS, and Dup15q; positive predictive and negative predictive values of 100% for PWS and AS; and a positive predictive value of 87.5% and negative predictive value of 100% for Dup15q. In the test data set of NBS samples from 16 579 infants, 92 had a positive test result using a methylation ratio cut-off of 3 standard deviations from the mean. Of these patients, 2 were confirmed to have PWS; 2, AS; and 1, maternal Dup15q. With the use of more conservative PWS- and AS-specific thresholds for positive calls from the validation data set, 9 positive NBS results were identified by MS-QMA in this cohort. The 2 PWS and 2 AS calls were confirmed by second-tier testing, but the 1 Dup15q case was not confirmed. Together, these results provided prevalence estimates of 1 in 8290 for both AS and PWS and 1 in 16 579 for maternal Dup15q, with positive predictive values for first-tier testing at 67.0% for AS, 33.0% for PWS, and 44.0% for combined detection of chromosome 15 imprinting disorders for the validation data set. Conclusions and Relevance: The findings of this diagnostic study suggest that it is feasible to screen for all chromosome 15 imprinting disorders using SNRPN methylation analysis, with 5 individuals identified with these disorders out of 16 579 infants screened.

A new test for autism spectrum disorder: Metabolic data from different cell types.

Experiments employing the Phenotype Mammalian Microarray (PM-M) technology were performed on lymphoblastoid cell lines (LCLs) from individuals with autism spectrum disorder (ASD) and age-matched controls. We used the custom-made PM-M plate designed to assess differential utilization of the amino acid tryptophan. Multiple parameters such as the sample size, incubation time, and cell concentration have been tested, leading to optimized protocols and minimized background noise by variable selection while controlling for false discoveries. The assay generated data based on the production of nicotinamide adenine dinucleotide (NADH) in the presence of different compounds containing tryptophan and showed clear differences between ASD and control samples.

Detection of a DNA Methylation Signature for the Intellectual Developmental Disorder, X-Linked, Syndromic, Armfield Type.

A growing number of genetic neurodevelopmental disorders are known to be associated with unique genomic DNA methylation patterns, called episignatures, which are detectable in peripheral blood. The intellectual developmental disorder, X-linked, syndromic, Armfield type (MRXSA) is caused by missense variants in FAM50A. Functional studies revealed the pathogenesis to be a spliceosomopathy that is characterized by atypical mRNA processing during development. In this study, we assessed the peripheral blood specimens in a cohort of individuals with MRXSA and detected a unique and highly specific DNA methylation episignature associated with this disorder. We used this episignature to construct a support vector machine model capable of sensitive and specific identification of individuals with pathogenic variants in FAM50A. This study contributes to the expanding number of genetic neurodevelopmental disorders with defined DNA methylation episignatures, provides an additional understanding of the associated molecular mechanisms, and further enhances our ability to diagnose patients with rare disorders.

Abnormalities in the genes that encode Large Amino Acid Transporters increase the risk of Autism Spectrum Disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a common neurodevelopmental disorder whose molecular mechanisms are largely unknown. Several studies have shown an association between ASD and abnormalities in the metabolism of amino acids, specifically tryptophan and branched-chain amino acids (BCAAs). METHODS: Ninety-seven patients with ASD were screened by Sanger sequencing the genes encoding the heavy (SLC3A2) and light subunits (SLC7A5 and SLC7A8) of the large amino acid transporters (LAT) 1 and 2. LAT1 and 2 are responsible for the transportation of tryptophan and BCAA across the blood-brain barrier and are expressed both in blood and brain. Functional studies were performed employing the Biolog Phenotype Microarray Mammalian (PM-M) technology to investigate the metabolic profiling in lymphoblastoid cell lines from 43 patients with ASD and 50 controls with particular focus on the amino acid substrates of LATs. RESULTS: We detected nine likely pathogenic variants in 11 of 97 patients (11.3%): three in SLC3A2, three in SLC7A5, and three in SLC7A8. Six variants of unknown significance were detected in eight patients, two of which also carrying a likely pathogenic variant. The functional studies showed a consistently reduced utilization of tryptophan, accompanied by evidence of reduced utilization of other large aromatic amino acids (LAAs), either alone or as part of a dipeptide. CONCLUSION: Coding variants in the LAT genes were detected in 17 of 97 patients with ASD (17.5%). Metabolic assays indicate that such abnormalities affect the utilization of certain amino acids, particularly tryptophan and other LAAs, with potential consequences on their transport across the blood barrier and their availability during brain development. Therefore, abnormalities in the LAT1 and two transporters are likely associated with an increased risk of developing ASD.

Decreased tryptophan metabolism in patients with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASDs) are relatively common neurodevelopmental conditions whose biological basis has been incompletely determined. Several biochemical markers have been associated with ASDs, but there is still no laboratory test for these conditions. METHODS: We analyzed the metabolic profile of lymphoblastoid cell lines from 137 patients with neurodevelopmental disorders with or without ASDs and 78 normal individuals, using Biolog Phenotype MicroArrays. RESULTS: Metabolic profiling of lymphoblastoid cells revealed that the 87 patients with ASD as a clinical feature, as compared to the 78 controls, exhibited on average reduced generation of NADH when tryptophan was the sole energy source. The results correlated with the behavioral traits associated with either syndromal or non-syndromal autism, independent of the genetic background of the individual. The low level of NADH generation in the presence of tryptophan was not observed in cell lines from non-ASD patients with intellectual disability, schizophrenia or conditions exhibiting several similarities with syndromal autism except for the behavioral traits. Analysis of a previous small gene expression study found abnormal levels for some genes involved in tryptophan metabolic pathways in 10 patients. CONCLUSIONS: Tryptophan is a precursor of important compounds, such as serotonin, quinolinic acid, and kynurenic acid, which are involved in neurodevelopment and synaptogenesis. In addition, quinolinic acid is the structural precursor of NAD+, a critical energy carrier in mitochondria. Also, the serotonin branch of the tryptophan metabolic pathway generates NADH. Lastly, the levels of quinolinic and kynurenic acid are strongly influenced by the activity of the immune system. Therefore, decreased tryptophan metabolism may alter brain development, neuroimmune activity and mitochondrial function. Our finding of decreased tryptophan metabolism appears to provide a unifying biochemical basis for ASDs and perhaps an initial step in the development of a diagnostic assay for ASDs.

Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders.

Autism spectrum disorders (ASDs) include three main conditions: autistic disorder (AD), pervasive developmental disorder, not otherwise specified (PDD-NOS), and Asperger syndrome. It has been shown that many genes associated with ASDs are involved in the neuroligin-neurexin interaction at the glutamate synapse: NLGN3, NLGN4, NRXN1, CNTNAP2, and SHANK3. We screened this last gene in two cohorts of ASD patients (133 patients from US and 88 from Italy). We found 5/221 (2.3%) cases with pathogenic alterations: a 106 kb deletion encompassing the SHANK3 gene, two frameshift mutations leading to premature stop codons, a missense mutation (p.Pro141Ala), and a splicing mutation (c.1820-4 G>A). Additionally, in 17 patients (7.7%) we detected a c.1304+48C>T transition affecting a methylated cytosine in a CpG island. This variant is reported as SNP rs76224556 and was found in both US and Italian controls, but it results significantly more frequent in our cases than in the control cohorts. The variant is also significantly more common among PDD-NOS cases than in AD cases. We also screened this gene in an independent replication cohort of 104 US patients with ASDs, in which we found a missense mutation (p.Ala1468Ser) in 1 patient (0.9%), and in 8 patients (7.7%) we detected the c.1304+48C>T transition. While SHANK3 variants are present in any ASD subtype, the SNP rs76224556 appears to be significantly associated with PDD-NOS cases. This represents the first evidence of a genotype-phenotype correlation in ASDs and highlights the importance of a detailed clinical-neuropsychiatric evaluation for the effective genetic screening of ASD patients.