Human Mutation

Background. Previous studies have established that heterozygous mutation for the p.R4810K variant can influence the severity of the clinical phenotype in patients with moyamoya disease (MMD) at disease onset. However, the relationship between the p.R4810K variant and the clinical phenotype of long-term unfavorable outcomes in Chinese pediatric patients remains unclear. Objectives. The primary aim of this study was to examine the association of heterozygous p.R4810K of RNF213 and long-term unfavorable outcomes after encephaloduroarteriosynangiosis (EDAS) in Chinese pediatric patients with MMD. Method. In this retrospective cohort study, we included 259 pediatric patients with MMD who possessed the known p.R4810K genotype. These individuals underwent EDAS along with genotyping analysis for p.R4810K via a TaqMan probe and the QuantStudio 6 Flex Real-Time PCR System. Subsequently, we evaluated their long-term outcomes. The variables we assessed were age at diagnosis, gender, p.R4810K genotypes, initial modified Rankin scale (mRS), clinical manifestations (such as hemorrhage and ischemia), posterior cerebral artery (PCA) involvement combined with angiographic stage, and their history of risk factors like hyperlipidemia and hyperhomocysteinemia. Furthermore, we scrutinized long-term unfavorable outcomes using both univariate analyses and multivariate logistic regression to identify independent predictive factors. Results. This study enrolled 259 Chinese pediatric patients with MMD, which included both newly and previously diagnosed cases, who underwent EDAS. The cohort comprised 130 male participants (50.19%) and 129 female participants (49.81%), with a median onset age of 8 years (median, IQR: 6-12 years). Among these patients, homozygous mutations were exceptionally rare, identified in only 4 individuals (1.54%), while the prevalence of heterozygous mutations was relatively higher, observed in 85 children (32.82%). The multivariate logistic regression showed that several factors were significantly associated with long-term unfavorable outcomes: older age at diagnosis (OR, 0.82 [95% CI, 0.7-0.96], ), onset with hematoma (OR, 12.76 [95% CI, 1.52-106.89], ), initial mRS (OR, 24.53 [95% CI, 6.51-92.41], ), perioperative infarction (OR, 22.16 [95% CI, 1.45-337.96], ), and infarction during follow-up (OR, 14.5 [95% CI, 2.04-103.12], ). Furthermore, the cumulative incidence of initial infarction suggested that pediatric patients with homozygous or heterozygous mutations typically present at a younger age and exhibit a higher incidence of initial infarction compared to those carrying wild-type genotypes. Conclusions. The study suggests that the p.R4810K variant is associated with the onset age of MMD in Chinese pediatric patients, potentially impacting long-term outcomes. Surprisingly low recurrent stroke rates were observed across all genotypes, including homozygous individuals for the pathogenic variant, indicating that nongenetic factors may also play a role in the course and outcomes of MMD in this population.

Recent advancements in the next-generation sequencing have illuminated the occurrence of multiple genetic diagnoses (MGD). While exome sequencing has provided insights, genome sequencing (GS), the most comprehensive diagnostic tool, remains underexplored for studying MGD prevalence. We retrospectively analyzed 1487 pediatric cases from our laboratory, employing GS to investigate the incidence of single definitive genetic diagnosis (SDD) and MGD in children suspected of having a genetic disease. Of these patients, 273 received at least one definitive diagnosis, including 245 with SDD (16.5%) and 28 with MGD (1.9%). Diagnostic yield was consistent across genders and unaffected by previous testing in SDD cases. Notably, prior testing significantly increased the diagnostic yield in MGD cases to 2.7% overall and 14.4% among diagnosed cases, compared to 1.1% for those with GS as a first-tier test. Age was a significant factor in diagnostic outcome for both SDD and MGD cases with neonates showing the highest diagnostic yield of 24.5% in SDD and a notably higher yield in MGD at 4.9%, representing 16.7% of the diagnosed cases. Of the 28 MGD cases, 17 exhibited distinct phenotypes, 9 had overlapping features, and 2 presented a mix, underscoring the genetic and phenotypic heterogeneity within this group. This study is the first to exclusively use GS to assess MGD prevalence. Our findings highlight the complexity of rare diseases and emphasize the importance of comprehensive, genome-level diagnostics. Clinicians must ensure that diagnoses fully account for the observed phenotypes to inform optimal therapeutic strategies and management.

Although around 6% of the world’s population is affected by rare diseases, only a small number of disease-modifying therapies are available. In recent years, antisense oligonucleotides (ASOs) have emerged as one option for the development of therapeutics for orphan diseases. In particular, ASOs can be utilized for individualized genetic treatments, addressing patients with a known disease-causing genetic variant, who would otherwise not be able to receive therapy. Careful prioritization of genetic variants amenable to an ASO approach is crucial to increase chances for successful treatments and reduce costs and time for drug development. At present, there is no consensus on how to systematically approach this selection procedure. Here, we present practical guidelines to evaluate disease-causing variants and standardize the process of selecting -of-1 cases. We focus on variants leading to a loss of function in monogenic disorders and consider which splice-switching ASO-mediated treatments are applicable in each case. To ease the understanding and application of our guidelines, we created a hypothetical transcript covering different pathogenic variants and explained their evaluation in detail. We support our recommendations with real-life examples and add further considerations to be applied to specific cases to provide a comprehensive framework for selecting eligible variants.

Routine gene panel analysis identifies pathogenic variants in clinically relevant genes. However, variants of unknown significance (VUSs) are commonly observed, many of which potentially have an impact on mRNA transcription and splicing. Several software programs attempt to predict the impact of variants on splicing and thus make it possible to select the variants for which it is important to study the effect on the transcripts. Transcript analysis is also necessary to show the tandem character of large duplications, and it can be useful for the search for deep intronic variants that are difficult to identify in a DNA panel. We analyzed 53 variants of unknown significance by targeted sequencing of 48 genes using RNA extracted from patient blood samples. RT-PCR and Sanger sequencing of patient mRNA or minigene monoallelic analysis was also carried out when necessary. For the 53 VUSs, 21 could be classified as likely neutral and 10 as pathogenic or likely pathogenic. Data are comprehensively presented for four variants: PTEN c.206+6T>G, MLH1 c.791-489_791-20del, BRCA2 c.68-8_68-7delinsAA, and MSH2 c.(1076+1_1077-1)_(1276+1_1277-1)dup. These four examples illustrate the usefulness of blood RNA panel sequencing in clinical oncogenetics to help classify VUSs with predicted splice effects. It could also be useful for characterizing large duplications and for detecting deep intronic variants with an impact on expressed transcripts.

Genetic investigations of people with speech and language disorders can provide windows into key aspects of human biology. Most genomic research into impaired speech development has so far focused on childhood apraxia of speech (CAS), a rare neurodevelopmental disorder characterized by difficulties with coordinating rapid fine motor sequences that underlie proficient speech. In 2001, pathogenic variants of FOXP2 provided the first molecular genetic accounts of CAS aetiology. Since then, disruptions in several other genes have been implicated in CAS, with a substantial proportion of cases being explained by high-penetrance variants. However, the genetic architecture underlying other speech-related disorders remains less well understood. Thus, in the present study, we used systematic DNA sequencing methods to investigate idiopathic speech delay, as characterized by delayed speech development in the absence of a motor speech diagnosis (such as CAS), a language/reading disorder, or intellectual disability. We performed genome sequencing in a cohort of 23 children with a rigorous diagnosis of idiopathic speech delay. For roughly half of the sample (ten probands), sufficient DNA was also available for genome sequencing in both parents, allowing discovery of de novo variants. In the thirteen singleton probands, we focused on identifying loss-of-function and likely damaging missense variants in genes intolerant to such mutations. We found that one speech delay proband carried a pathogenic frameshift deletion in SETD1A, a gene previously implicated in a broader variable monogenic syndrome characterized by global developmental problems including delayed speech and/or language development, mild intellectual disability, facial dysmorphisms, and behavioural and psychiatric symptoms. Of note, pathogenic SETD1A variants have been independently reported in children with CAS in two separate studies. In other probands in our speech delay cohort, likely pathogenic missense variants were identified affecting highly conserved amino acids in key functional domains of SPTBN1 and ARF3. Overall, this study expands the phenotype spectrum associated with pathogenic SETD1A variants, to also include idiopathic speech delay without CAS or intellectual disability, and suggests additional novel potential candidate genes that may harbour high-penetrance variants that can disrupt speech development.

Pyruvate dehydrogenase complex deficiency (PDCD) is a defect of aerobic carbohydrate metabolism that causes neurological disorders with varying degrees of severity. We report the clinical, biochemical, and molecular findings in patients with primary and secondary PDCD caused by novel atypical genetic variants. Whole-genome sequencing (WGS) identified the synonymous variants c.447A>G, p.(Lys149=) and c.570C>T, p.(Cys190=) in pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), the deep intronic variants c.1023+2267G>A and c.1023+2302A>G in pyruvate dehydrogenase complex component X (PDHX), and c.185+15054G>A in thiamine pyrophosphokinase (TPK1). Analysis by Sanger and RNA sequencing of cDNA from patient blood and/or cultured fibroblasts showed that the synonymous variants in PDHA1 lead to aberrant splicing and skipping of exons 5 and 5-6 in one of the patients and transcripts lacking exon 6 in the other. The deep intronic variants in PDHX and TPK1 lead to insertion of intronic sequence in the corresponding transcripts. The splice defects in PDHA1 were more pronounced in cultured fibroblasts than in blood. Our findings expand the spectrum of pathogenic variants causing PDCD and highlight the importance of atypical variants leading to aberrant splicing. The severity of the splice defects and resulting biochemical dysfunction varied between tissues, stressing the importance of performing biochemical and transcript analysis in affected tissues. The two males with hemizygous synonymous PDHA1 variants have a mild phenotype and higher PDH enzyme activity than expected, which is consistent with aberrant but leaky splicing with a proportion of the transcripts remaining correctly spliced.