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Pompe disease (PD) is a rare autosomal recessive lysosomal disorder caused by loss‐of‐function of the α‐glucosidase (GAA) gene. The deficient GAA enzyme activity may result in potential life‐threatening muscle weakness, thus requiring a rapid diagnosis to initiate therapeutic interventions. In this large retrospective study, we analyzed 30.836 PD suspect samples from 57 countries using a two‐step approach utilizing dried blood spots (DBSs): biochemical testing of GAA activity followed by complementary genetic sequencing of GAA in biochemically conspicuous cases. Of these 30.836 samples, 2% (n = 639) were excluded; accordingly, this study consisted of 30.193 cases. Biochemical testing of GAA enzyme activity showed normal values in 28.354 (93.90%) and enzyme activity below the cut‐off in 1843 (6.10%) cases. These biochemically suspicious cases were genetically analyzed. We identified 723 Pompe cases with 283 different GAA alterations, and 98 variants have been unpublished so far. The most common variant was the splice variant c.‐32‐13T>G (IVS1). Looking at the IVS1‐genotype, the majority was compound heterozygous (n = 169) and identified in late‐onset cases (n = 162). Comparison of early‐ versus late‐onset cases to evaluate whether certain genotypes correlate with the age of onset revealed that homozygosity was predominantly found in infantile (85.65%) and compound heterozygosity in late‐onset (76.9%) cases. Analysis of homozygous cases revealed 61% nonsense variants in the early stages and 87% missense variants in the late stages. Mapping of disease‐associated (homozygous) missense variants to functional GAA protein domains showed that missense variants were found throughout GAA, but we identified enrichment in the catalytic domain. A strict genotype–phenotype correlation cannot be established; nevertheless, a phenotypic implication of some GAA variants could be drawn (e.g., c.896T>C/p.L299P, c.2015G>A/p.R672Q, and c.‐32‐13T>G). The combined enzyme activity and genetic testing from DBS cards can reliably identify PD and significantly accelerate diagnosis. We identified new genetic variants that contribute to the spectrum of pathogenic variants of the GAA gene. [ABSTRACT FROM AUTHOR]

Enlarged vestibular aqueduct (EVA) is a frequently occurring inner ear malformation that associates with sensorineural hearing loss (SNHL), with SLC26A4 being the responsible gene. Based on multiplex PCR enrichment and sequencing of the exonic and flanking regions of the SLC26A4 gene, we developed a panel specifically for EVA and found that up to 95% of EVA patients in our Chinese cohorts carried biallelic SLC26A4 pathogenic variants (M2). In this study, we tried to investigate the genetic etiology of 13 previously undiagnosed EVA patients with monoallelic (M1) or none (M0) SLC26A4 variant using a stepwise approach, including copy number variation (CNV) analysis of multiplex PCR enrichment and next‐generation sequencing data, single‐molecule real‐time (SMRT) sequencing of the whole SLC26A4 gene, whole exome sequencing (WES), and whole genome sequencing (WGS). CNV analysis revealed deletions in Exons 1–3, Exons 5–6, and Exons 9–10 of the SLC26A4 gene in seven patients, and SMRT sequencing identified the same heterozygous deep intronic variant (NM_000441.2:c.304+941C>T) in two patients, resulting in a final diagnosis in 9/13 patients. Notably, the variants of Exons 9–10 deletion and c.304+941C>T have not been reported previously. We further showed that the variant c.304+941C>T led to the exonization of partial AluSz6 element (126 bp) where the variant is located through sequencing of the mRNA extracted from the blood of a heterozygous variant carrier. In conclusion, our stepwise approach improved the diagnosis rate of EVA, expanded the mutational spectrum of the SLC26A4 gene, and highlighted the contribution of exonic deletions and deep intronic variants to EVA. [ABSTRACT FROM AUTHOR]

Chronic pancreatitis (CP) is a complex disease with genetic and environmental factors at play. Through trio exome sequencing, a de novo SEC16A frameshift variant in a Chinese teenage CP patient is identified. Subsequent targeted next‐generation sequencing of the SEC16A gene in 1,061 Chinese CP patients and 1,196 controls reveals a higher allele frequency of rare nonsynonymous SEC16A variants in patients (4.90% vs 2.93%; odds ratio [OR], 1.71; 95% confidence interval [CI], 1.26–2.33). Similar enrichments are noted in a French cohort (OR, 2.74; 95% CI, 1.67–4.50) and in a biobank meta‐analysis (OR, 1.16; 95% CI, 1.04–1.31). Notably, Chinese CP patients with SEC16A variants exhibit a median onset age 5 years earlier than those without (40.0 vs 45.0; p = 0.012). Functional studies using three CRISPR/Cas9‐edited HEK293T cell lines show that loss‐of‐function SEC16A variants disrupt coat protein complex II (COPII) formation, impede secretory protein vesicles trafficking, and induce endoplasmic reticulum (ER) stress due to protein overload. Sec16a+/− mice, which demonstrate impaired zymogen secretion and exacerbated ER stress compared to Sec16a+/+, are further generated. In cerulein‐stimulated pancreatitis models, Sec16a+/− mice display heightened pancreatic inflammation and fibrosis compared to wild‐type mice. These findings implicate a novel pathogenic mechanism predisposing to CP. [ABSTRACT FROM AUTHOR]

Whole exome sequencing (WES) has become an increasingly common technique for identifying the genetic cause of Mendelian genetic diseases. However, it may fail to detect the complex regions of the genome. Here, we investigated the genetic etiology of a pedigree with autosomal dominant polycystic kidney disease (ADPKD) using a combination of WES, multiplex ligation‐dependent probe amplification (MLPA), Sanger sequencing, and long‐read sequencing (LRS). Initially, WES of the proband revealed a heterozygous variant c.7391G>C in PKD1 Exon 18, along with a heterozygous deletion of the 17th and 18th exons of PKD1 detected by exome‐based copy number variation (CNV) analysis. MLPA confirmed the PKD1 heterozygous deletion of Exon 18. Except for c.7391G>C, Sanger sequencing identified four other heterozygous variants (c.7278T>C, c.7288C>T, c.7344C>G, and c.7365C>T) in Exon 18 of PKD1. Subsequently, LRS uncovered seven clustered substitution variants (c.7209+28C>T, c.7210‐16C>T, c.7278T>C, c.7288C>T, c.7344C>G, c.7365C>T, and c.7391G>C), with six of them omitted by WES due to interference from PKD1 pseudogenes. Combining LRS results with cosegregation of the pedigree analysis, we found these variants were in cis and converted from PKD1 pseudogenes, covering a region of at least 282 bp. Notably, the paralogous sequence variants of c.7288C>T introduced a premature stop codon of PKD1, leading to a function loss, and were classified as pathogenic (PVS1+PS4+PM2) according to the ACMG/AMP guideline. Our study highlights the limitations of WES/MLPA and the importance of utilizing complementary tools like LRS for comprehensive variant detection in PKD1. [ABSTRACT FROM AUTHOR]

D-amphetamine has been used to enhance cognitive performance over the last few decades. Due to the rapid absorption after administration, d-amphetamine shows narrow effective window and severe abuse potential. Lisdexamfetamine, a prodrug of d-amphetamine, reduces the magnitude of plasma d-amphetamine concentration and prolongs the action duration when compared with immediate-release d-amphetamine at equimolar doses. However, the differences of these two drugs, which produce distinct pharmacokinetic characteristics, in cognition improvement still unclear. In present study, we compared the effects of d-amphetamine (i.p) and lisdexamfetamine (p.o) at equimolar doses (0.2, 0.5, 1.5, 4.5, and 13.5 mg/kg of d-amphetamine base) on locomotion, spatial working memory and recognition memory in rats. Given the crucial involvement of dopamine neurotransmitter system within the medial prefrontal cortex (mPFC) in cognitive processing, microdialysis was conducted to profile the difference in neurochemical characteristics between the two drugs. In our results, d-amphetamine ranges from 0.5 to 1.5 mg/kg significantly increased locomotor activity. However, d-amphetamine ranges from 0.2 to 13.5 mg/kg failed to improve spatial working memory and recognition memory in Y-maze-based spontaneous alternation and two-trial delayed alternation tasks of rats, respectively. In contrast, lisdexamfetamine with 4.5 mg/kg significantly increased the locomotion and improved both spatial working and recognition memory. Further, microdialysis showed that lisdexamfetamine induced lower magnitude and longer duration of extracellular dopamine increase than that of d-amphetamine. These results suggest that lisdexamfetamine was more effective than d-amphetamine in improving spatial cognitive performance, which was attributed to the steady and lasting dopamine release pattern within the mPFC.

Preimplantation embryonic developmental arrest (EDA) is a common cause of unexplained female infertility. Genetic factors are believed to be one of the primary causes contributing to EDA. In this study, we identify four novel compound heterozygous mutations in TLE6 and NLRP5, in two infertile female patients experiencing recurrent EDA, using whole‐exome sequencing. Functional analysis revealed that the two splicing mutations in TLE6 (c.541+2dupT) and NLRP5 (c.2957+4A>G) resulted in aberrant RNA splicing, leading to abnormal truncations of the corresponding proteins. In vitro experiments further validated that a missense mutation in NLRP5 led to increased mRNA and protein expression levels compared to wild type, when transfected into HEK293T cells. Immunofluorescence analysis confirmed the decay of the expression of TLE6 protein. Additionally, RNA sequencing results revealed significantly higher expression levels of some maternal genes in mutated embryos with TLE6 mutations, possibly suggesting the disrupted clearance of maternal mRNA and the failure of embryo genome activation. These results highlight the role of biallelic recessive effects associated with TLE6 and NLRP5 variants in embryonic development, thereby widening the scope of the genetic landscape. [ABSTRACT FROM AUTHOR]