UNASSIGNED: Disheveled, EGL-10, and pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a component of GTPase-activating protein (GAP) activity toward the RAG complex 1 (GATOR1) protein, which is an inhibitor of the amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. GATOR1 complex variations were reported to correlate with familial focal epilepsy with variable foci (FFEVF). With the wide application of whole exome sequencing (WES), more and more variations in DEPDC5 were uncovered in FFEVF families.
UNASSIGNED: A family with a proband diagnosed with familial focal epilepsy with variable foci (FFEVF) was involved in this study. Whole exome sequencing (WES) was performed in the proband, and Sanger sequencing was used to confirm the variation carrying status of the family members. Mini-gene splicing assay was performed to validate the effect on the alternative splicing of the variation.
UNASSIGNED: A novel variant, c.1217 + 2T>A, in DEPDC5 was identified by WES in the proband. This splicing variant that occurred at the 5\' end of intron 17 was confirmed by mini-gene splicing assays, which impacted alternative splicing and led to the inclusion of an intron fragment. The analysis of the transcribed mRNA sequence indicates that the translation of the protein is terminated prematurely, which is very likely to result in the loss of function of the protein and lead to the occurrence of FFEVF.
UNASSIGNED: The results suggest that c.1217 + 2T>A variations in DEPDC5 might be the genetic etiology for FFEVF in this pedigree. This finding expands the genotype spectrum of FFEVF and provides new etiological information for FFEVF.

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Parapoxviruses (PPV) of animals are spread worldwide. While the Orf virus (ORFV) species is a molecularly well-characterized prototype pathogen of small ruminants, the genomes of virus species affecting large ruminants, namely Bovine papular stomatitis virus (BPSV) and Pseudocowpox virus (PCPV), are less well known. Using Nanopore sequencing we retrospectively show the whole genome sequences (WGS) of six BPSV, three PCPV isolates and an attenuated ORFV strain, originating from different geographic locations. A phylogenetic tree shows that the de novo assembled genomes belong to PPV species including WGS of reference PPV. Remarkably, Nanopore sequencing allowed the molecular resolution of inverted terminal repeats (ITR) and the hairpin loop within the de novo assembled WGS. Additionally, peculiarities regarding map location of two genes and the heterogeneity of a genomic region were noted. Details for the molecular variability of an interferon response modulatory gene (ORF116) and the PCPV specificity of gene 073.5 are reported. In summary, WGS gained by Nanopore sequencing allowed analysis of complete PPV genomes and confident virus species attribution within a phylogenetic tree avoiding uncertainty of limited gene-based diagnostics. Nanopore-based WGS provides robust comparison of PPV genomes and reliable identity determination of new Poxviruses.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a type of non-motile ciliopathy. To date, 26 genes have been reported to be associated with BBS. However, BBS is genetically heterogeneous, with significant clinical overlap with other ciliopathies, which complicates diagnosis. Disability and mortality rates are high in BBS patients; therefore, it is urgent to improve our understanding of BBS. Thus, our study aimed to describe the genotypic and phenotypic spectra of BBS in China and to elucidate genotype-phenotype correlations.
METHODS: Twenty Chinese patients diagnosed with BBS were enrolled in this study. We compared the phenotypes of Chinese BBS patients in this study with those from other countries to analyze the phenotypic differences across patients worldwide. In addition, genotype-phenotype correlations were described for our cohort. We also summarized all previously reported cases of BBS in Chinese patients (71 patients) and identified common and specific genetic variants in the Chinese population.
RESULTS: Twenty-eight variants, of which 10 are novel, in 5 different BBS-associated genes were identified in 20 Chinese BBS patients. By comparing the phenotypes of BBSome-coding genes (BBS2,7,9) with those of chaperonin-coding genes (BBS10,12), we found that patients with mutations in BBS10 and 12 had an earlier age of onset (1.10 Vs. 2.20, p < 0.01) and diagnosis (4.64 Vs. 13.17, p < 0.01), whereas patients with mutations in BBS2, 7, and 9 had a higher body mass index (28.35 Vs. 24.21, p < 0.05) and more vision problems (p < 0.05). Furthermore, in 91 Chinese BBS patients, mutations were predominant in BBS2 (28.89%) and BBS7 (15.56%), and the most frequent variants were in BBS2: c.534 + 1G > T (10/182 alleles) and BBS7: c.1002delT (7/182 alleles), marking a difference from the genotypic spectra of BBS reported abroad.
CONCLUSIONS: We recruited 20 Chinese patients with BBS for genetic and phenotypic analyses, and identified common clinical manifestations, pathogenic genes, and variants. We also described the phenotypic differences across patients worldwide and among different BBS-associated genes. This study involved the largest cohort of Chinese patients with BBS, and provides new insights into the distinctive clinical features of specific pathogenic variants.

Delaying childbearing age has become a trend in modern times, but it has also led to a common challenge in clinical reproductive medicine-diminished ovarian reserve (DOR). Since the mechanism behind DOR is unknown and its clinical features are complex, physicians find it difficult to provide targeted treatment. Many factors affect ovarian reserve function, and existing studies have shown that genetic variants, upstream regulatory genes, and changes in protein expression levels are present in populations with reduced ovarian reserve function. However, existing therapeutic regimens often do not target the genetic profile for more individualized treatment. In this paper, we review the types of genetic variants, mutations, altered expression levels of microRNAs, and other related factors and their effects on the regulation of follicular development, as well as altered DNA methylation. We hope this review will have significant implications for the future treatment of individuals with reduced ovarian reserve.

OBJECTIVE: Teratozoospermia is the main pathogenic factor of male infertility. However, the genetic etiology of teratozoospermia is largely unknown. This study aims to clarify the relationship between novel variations in TENT5D and teratozoospermia in infertile patients.
METHODS: Two infertile patients were enrolled. Routine semen analysis of patients and normal controls was conducted with the WHO guidelines. Whole-exome sequencing (WES) was conducted to identify pathogenic variants in the two patients. Morphology and ultrastructure analysis of spermatozoa in the two patients was determined by Papanicolaou staining, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The functional effect of the identified variants was analyzed by immunofluorescence staining and western blotting. The expression of TENT5D in different germ cells was detected by immunofluorescence staining.
RESULTS: Two new hemizygous variations, c.101C > T (p.P34L) and c.125A > T (p.D42V), in TENT5D were detected in two patients with male infertility. Morphology analysis showed abnormalities in spermatozoa morphology in the two patients, including multiple heads, headless, multiple tails, coiled, and/or bent flagella. Ultrastructure analysis showed that most of the spermatozoa exhibited missing or irregularly arranged \'9+2\' structures. Further functional experiments confirmed the abrogated TENT5D protein expression in patients. In addition, both p.P34L and p.D42V substitutions resulted in a conformational change of the TENT5D protein. We precisely analyzed the subcellular localization of TENT5D in germ cells in humans and mice. And we found that TENT5D was predominantly detected in the head and flagellum of elongating spermatids and epididymal spermatozoa.
CONCLUSIONS: Our results showed further evidence of a relationship between TENT5D mutation and human male infertility, providing new genetic insight for use in the diagnosis and treatment of male infertility.

The extra copy of the methyl-CpG-binding protein 2 (MeCp2) gene causes MeCP2 duplication syndrome (MDS), a neurodevelopmental disorder characterized by intellectual disability and autistic phenotypes. However, the disturbed microbiome and metabolic profiling underlying the autistic-like behavioral deficits of MDS are rarely investigated. Here we aimed to understand the contributions of microbiome disruption and associated metabolic alterations, especially the disturbed neurotransmitters in MDS employing a transgenic mouse model with MeCP2 overexpression. We analyzed metabolic profiles of plasma, urine, and cecum content and microbiome profiles by both 16 s RNA and shotgun metagenomics sequence technology. We found the decreased levels of Firmicutes and increased levels of Bacteroides in the single MeCP2 gene mutation autism-like mouse model, demonstrating the importance of the host genome in a selection of microbiome, leading to the heterogeneity characteristics of microbiome in MDS. Furthermore, the changed levels of several neurotransmitters (such as dopamine, taurine, and glutamate) implied the excitatory-inhibitory imbalance caused by the single gene mutation. Concurrently, a range of microbial metabolisms of aromatic amino acids (such as tryptophan and phenylalanine) were identified in different biological matrices obtained from MeCP2 transgenic mice. Our investigation revealed the importance of genetic variation in accounting for the differences in microbiomes and confirmed the bidirectional regulatory axis of microbiota-gut-brain in studying the role of microbiome on MDS, which could be useful in deeply understanding the microbiome-based treatment in this autistic-like disease.

OBJECTIVE: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4).
METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children\'s Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types.
RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations.
CONCLUSIONS: ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.
目的: 探讨丁酰基肉碱（C4）代谢异常新生儿的基因型和生化表型特征。方法: 收集2018年1月至2023年6月在浙江大学医学院附属儿童医院经串联质谱法筛查单纯C4增高的120例新生儿初筛和召回复查的C4、C4/C3检测数据，并换算为C4增高倍数。采用液相捕获技术靶向捕获酰基辅酶A脱氢酶8（ACAD8）和短链酰基辅酶A脱氢酶（ACADS）基因的外显子及邻近50 bp区域，通过高通量测序和生物信息学分析获取基因变异信息，参考美国医学遗传学与基因组学学会分类标准进行致病性评估。采用威尔科克森秩和检验分析不同基因型新生儿C4增高倍数的差异。结果: 共检出32种ACAD8基因变异型，其中7种变异型未见报道；检出41种ACADS基因变异型，其中17种变异型未见报道。ACAD8双等位基因变异39例，ACAD8单等位基因变异3例，ACADS双等位基因变异34例，ACADS单等位基因变异36例，ACAD8和ACADS双基因变异5例。ACAD8双等位基因变异组、ACADS双等位基因变异组C4增高倍数初筛值和召回值均高于ACADS单等位基因变异组（均P<0.01），且ACAD8双等位基因变异组C4增高倍数初筛值和召回值较ACADS双等位基因变异组更高（均P<0.01）。所有携带ACAD8或ACADS双等位基因变异新生儿的初筛C4增高倍数均大于1.5；而仅有25%（9/36）携带ACADS单等位基因变异的新生儿初筛C4增高倍数大于1.5。结论: ACAD8和（或）ACADS基因变异是浙江地区新生儿C4增高的主要遗传学原因，其变异型具有高度异质性。双等位基因变异者C4水平高于单等位基因变异者。常规串联质谱法新生儿筛查结果为单纯的C4增高时，其“筛查切值”可以适当提升。.

OBJECTIVE: To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022.
METHODS: A total of 11 922 318 newborns were screened from September 1999 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by a fluorescence method and blood phenylalanine (Phe) levels were measured by fluorescence method or tandem mass spectrometry. TSH≥9 μIU/mL was considered positive for CH, while Phe>120 μmol/L and/or Phe/Tyr ratio>2.0 were considered positive for HPA. The positive newborns in screening were recalled, and the gene variations were detected by high-throughput sequencing and MassARRAY tests.
RESULTS: The overall neonatal screening rate during 1999-2022 was 89.41% (11 922 318/13 333 929) and the screening rate was increased from 6.46% in 1999 to 100.0% in 2022. A total of 8924 cases of CH were diagnosed among screened newborns with an incidence rate of 1/1336. A total of 563 cases of HPA were diagnosed, including 508 cases of classic phenylketonuria (cPKU) and 55 cases of tetrahydrobiopterin deficiency (BH4D), with an incidence rate of 1/21 176. Ninety-seven out of 8924 cases of CH underwent genetic analysis. Gene mutations were detected in 9 CH related genes, the highest frequency mutations were found in DUOX2 gene (69.0%) with c.3329G>A (p.R1110Q) (18.2%) and c.1588A>T (p.K530X) (17.3%) as the hotspot mutations. There were 81 PAH gene variants detected in a total of 250 cases of cPKU, and c728G>A (p.R243Q) (24.4%), c.721C>T (p.R241C) (15.0%) were the hotspot mutations. Meanwhile 7 novel variants in PAH gene were detected: c.107C>A (p.S36*), c.137G>T (p.G46V), c.148A>G(p.K50E), c.285C>T (p.I95I), c.843-10delTTCC, exon4-7del and c.1066-2A>G. There were 12 PTS gene variants detected in 36 cases of BH4D, and c.259C>T (p.P87S) (31.9%) was the hotspot mutation.
CONCLUSIONS: The incident of CH has increased from 1999 to 2022 in Zhejiang province, and it is higher than that of national and global levels; while the incidence of HPA is similar to the national average. DUOX2 gene variation is the most common in CH patients; c.728G>A (p.R243Q) is the hotspot mutation in cPKU patients, while c.259C>T (p.P87S) is the hotspot mutation in BH4D patients.
目的: 了解浙江省新生儿先天性甲状腺功能减退症（CH）及高苯丙氨酸血症（HPA）的筛查及发病情况。方法: 收集1999年9月至2022年12月浙江省新生儿疾病筛查中心11 922 318名新生儿血促甲状腺素（TSH）及苯丙氨酸（Phe）检测的资料。其中，血TSH浓度采用荧光法检测，血Phe浓度采用荧光法或串联质谱法检测。TSH在9 μIU/mL及以上的新生儿为CH筛查阳性，血Phe浓度超过120 μmol/L和（或）血Phe与酪氨酸（Tyr）比值大于2.0为HPA筛查阳性，召回复查。采用高通量测序和MassARRAY技术检测基因变异。结果: 1999—2022年共筛查新生儿11 922 318名，总筛查率为89.41%，筛查率由1999年6.46%提升至2022年的100.00%。检出CH患儿8924例，患病率为1/1336，检出HPA患儿563例，患病率为1/21 176，其中508例为经典型苯丙酮尿症（cPKU），55例为四氢生物蝶呤缺乏症（BH4D）。97例CH患儿完善基因检测，检出9种CH相关基因，双氧化酶2（DUOX2）基因检出频率最高（69.0%），其中c.3329G>A（p.R1110Q）（18.2%）和c.1588A>T（p.K530X）（17.3%）为热点突变。250例cPKU患者中检出81种苯丙氨酸羟化酶（PAH）基因变异，其中c728G>A（p.R243Q）（24.4%）和c.721C>T（p.R241C）（15.0%）为热点突变，7种为新发变异：c.107C>A（p.S36*）、c.137G>T（p.G46V）、c.148A>G（p.K50E）、c.285C>T（p.I95I）、c.843-10delTTCC、exon4-7del和c.1066-2A>G。36例BH4D患者中检出12种6-丙酮酰四氢蝶呤合成酶（PTS）基因变异，热点突变为c.259C>T（p.P87S）（31.9%）。结论: 浙江省新生儿筛查率逐年升高，CH发病率明显增高，HPA发病率与全国平均水平接近。DUOX2基因变异为CH患儿最常见基因变异，c728G>A（p.R243Q）为cPKU患者的热点突变，c.259C>T（p.P87S）为BH4D患者的热点突变。.

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction.