Objective: To investigate the pathogenic mechanism and clinical characteristics of the novel splicing variant of ATP-binding cassette subfamily B member 4 (ABCB4) and provide a basis for subsequent genetic diagnosis. Methods: The clinical data of a 5-year-old child with cholestatic liver disease admitted to the Beijing Children\'s Hospital of Capital Medical University was retrospectively analyzed. The pathogenic variations were detected by whole exome sequencing and verified by Sanger sequencing, and bioinformatics was used to predict the pathogenicity of the mutation sites. Possible pathogenic variations were verified in vitro by Minigene assay. The clinical outcome was followed after discharge from hospital. Results: The 5-year-old boy had developed cholestasis at the age of 11 months. His physical examination showed obvious enlargement of the liver and spleen. Cholestatic cirrhosis was diagnosed by liver function tests, abdominal ultrasonography, liver biopsy and pathology. The results of genetic analysis showed that the patient was a complex heterozygote of the ABCB4 gene, with a pathogenic mutation c.2860G>A and a novel mutation c.2065-8T>G, derived from the mother and father respectively. The conservative prediction of the c.2065-8T>G site showed that this region was highly conserved and may affect splicing. Minigene assay results confirmed that the c.2065-8T>G mutation resulted in a 7 bp retention of intron 16 in the mature mRNA. In the absence of nonsense-mediated mRNA decay, the amino acid frameshift forms a truncated protein, which is represented by p.Glu689ValfsTer19. The patient was diagnosed as progressive familial intrahepatic cholestasis type 3 (PFIC3) and treated with ursodeoxycholic acid (UDCA). His clinical symptoms improved during 18 months of follow-up. Conclusions: The c.2065-8T>G variant is confirmed to affect the splicing process and exhibits complex heterozygosity with c.2860G>A, which is identified as the cause of the disease. PFIC3 children with this variant showed cholestatic liver disease as the main manifestation with a slow progression and was sensitive to treatment with UDCA.
目的： 探讨ATP结合盒转运蛋白B4（ABCB4）新型剪接变异的分子致病机制及临床特点。 方法： 回顾性总结首都医科大学附属北京儿童医院收治的1例胆汁淤积性肝病患儿的临床资料，采用全外显子组测序检测致病突变并进行Sanger测序验证，通过生物信息学预测突变位点的致病性，通过mRNA异常剪接的Minigene体外验证实验对可能致病突变进行体外功能验证，并随访患儿出院后的临床转归。 结果： 患儿　男，5岁，11月龄出现胆汁淤积，体格检查显示肝脾明显肿大，肝功能、腹部超声及肝脏病理等检查提示胆汁淤积性肝硬化。基因分析结果显示患儿为ABCB4基因致病性突变c.2860G>A和新发突变c.2065-8T>G的复合杂合子，突变分别来源于其父母，对c.2065-8T>G位点进行保守性预测，显示该区域高度保守并可能影响剪接。Minigene实验结果证实c.2065-8T>G突变导致内含子16滞留7 bp序列在成熟的mRNA中，在未发生无义介导的mRNA降解的情况下，氨基酸移码形成截短蛋白（p.Glu689ValfsTer19），最终确诊为进行性家族性肝内胆汁淤积症3型（PFIC3），予熊去氧胆酸（UDCA）等药物治疗，在18个月的随访期内，患儿的临床症状有所改善。 结论： ABCB4基因c.2065-8T>G位点被证实影响剪接过程，与c.2860G>A构成复合杂合子，确定为PFIC3的致病原因，携带此突变的PFIC3患儿以胆汁淤积性肝病为主要表现，疾病进展相对缓慢，对UDCA治疗敏感。.

BACKGROUND: TBX6, a member of the T-box gene family, encodes the transcription factor box 6 that is critical for somite segmentation in vertebrates. It is known that the compound heterozygosity of disruptive variants in trans with a common hypomorphic risk haplotype (T-C-A) in the TBX6 gene contribute to 10% of congenital scoliosis (CS) cases. The deletion of chromosome 17q12 is a rare cytogenetic abnormality, which often leads to renal cysts and diabetes mellitus. However, the affected individuals often exhibit clinical heterogeneity and incomplete penetrance.
METHODS: We here present a Chinese fetus who was shown to have CS by ultrasound examination at 17 weeks of gestation. Trio whole-exome sequencing (WES) was performed to investigate the underlying genetic defects of the fetus. In vitro functional experiments, including western-blotting and luciferase transactivation assay, were performed to determine the pathogenicity of the novel variant of TBX6.
RESULTS: WES revealed the fetus harbored a compound heterozygous variant of c.338_340del (p.Ile113del) and the common hypomorphic risk haplotype of the TBX6 gene. In vitro functional study showed the p.Ile113del variant had no impact on TBX6 expression, but almost led to complete loss of its transcriptional activity. In addition, we identified a 1.85 Mb deletion on 17q12 region in the fetus and the mother. Though there is currently no clinical phenotype associated with this copy number variation in the fetus, it can explain multiple renal cysts in the pregnant woman.
CONCLUSIONS: This study is the first to report a Chinese fetus with a single amino acid deletion variant and a T-C-A haplotype of TBX6. The clinical heterogeneity of 17q12 microdeletion poses significant challenges for prenatal genetic counseling. Our results once again suggest the complexity of prenatal genetic diagnosis.

Objective: The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored. Methods: The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (β3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and β3 in platelets were analyzed by Western blot. Results: Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and β3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5\'SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the β-propeller domain of the p.S160-S192 deletion lost two β-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a β chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of β3 was 11.36% of the normal level. Conclusion: The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.
目的： 对一个ITGA2B基因复合杂合突变导致的遗传性血小板无力症家系进行表型及基因型研究，并探索其分子致病机制。 方法： 使用二磷酸腺苷、胶原、肾上腺素、花生四烯酸及瑞斯托霉素等诱聚剂进行血小板聚集试验，检测先证者及家系成员的血小板聚集率。通过流式细胞术检测血小板表面CD41（αⅡb）、CD61（β3）、CD42b（GPⅠb）的表达。采用基因测序技术进行基因鉴定。利用RT-PCR检测ITGA2B基因mRNA剪接情况，qRT-PCR检测ITGA2B基因mRNA相对水平。生物信息学分析评估突变位点的致病性及对蛋白结构和功能的影响。通过Western blot检测分析血小板总αⅡb、β3的表达。 结果： 除瑞斯托霉素外其他4种诱聚剂均无法使先证者血小板聚集。流式细胞术检测先证者血小板表面αⅡb的表达仅为0.25%，β3弱表达为9.76%，而GPⅠb表达相对正常，其余家系成员膜糖蛋白表达基本正常。基因测序结果显示先证者存在ITGA2B基因c.480C>G与c.2929C>T复合杂合突变，其中c.480C>G突变遗传自其母亲，c.2929C>T遗传自其父亲。RT-PCR及测序结果表明c.480C>G突变导致先证者及其母亲发生c.476G-574A（p.S160-S192）共99个碱基缺失的mRNA剪接。qRT-PCR检测发现c.2929C>T突变导致先证者及其父亲ITGA2B基因mRNA水平减低。生物信息学分析提示c.480C>G突变形成了与hnRNP A1蛋白结合序列，产生了5\'SS剪接位点。αⅡb亚基的蛋白三维结构模型显示，p.S160-S192缺失的β-propeller结构域第2 blade缺失两条β链和一个α螺旋；c.2929C>T无义突变使得翻译提前终止产生p.R977-E1039缺失的截短型蛋白，包括胞质域（CD）、跨膜域（TM）以及胞外Calf-2结构域一条β链的缺失。Western blot检测先证者血小板总αⅡb表达缺失、β3的相对表达量为正常人的11.36%。 结论： ITGA2B基因第4外显子c.480C>G与第28外显子c.2929C>T的复合杂合突变是本家系遗传性血小板无力症的致病原因。.

OBJECTIVE: To explore the clinical features and genetic basis for a fetus featuring Rhizomelic skeletal dysplasia.
METHODS: A fetus diagnosed at the Reproductive and Genetic Center of Suzhou Municipal Hospital in November 2020 was selected as the study subject. Whole exome sequencing (WES) was carried out for the fetus and its parents. Candidate variants were verified by Sanger sequencing. Peripheral blood smears of both parents were also examined.
RESULTS: The fetus was found to have a small chest and short limbs, which had suggested skeletal dysplasia. Genetic testing revealed that the fetus has harbored compound heterozygous variants of the LBR gene, including a paternally derived c.1687+1G>A and a maternally derived c.1757G>A (p.Arg586His). The blood smear of the father showed Pelger-Huet anomaly with hyposegmentation of neutrophil nuclei, while the neutrophils of the mother appeared to be normal. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP), the c.1757G>A (p.Arg586His) variant was classified as likely pathogenic (PM3_Strong+PM2_Supporting+PP3), and so was the c.1687+1G>A variant (PVS1-Moderate+PM3+PM2-Supporting+PP4).
CONCLUSIONS: The compound heterozygous variants of the LBR gene probably underlay the pathogenesis of skeletal dysplasia in this fetus.

Hereditary breast and ovarian cancer (HBOC) syndrome is a genetic condition that increases the risk of breast cancer by 80% and that of ovarian cancer by 40%. The most common pathogenic variants (PVs) causing HBOC occur in the BRCA1 gene, with more than 3850 reported mutations in the gene sequence. The prevalence of specific PVs in BRCA1 has increased across populations due to the effect of founder mutations. Therefore, when a founder mutation is identified, it becomes key to improving cancer risk characterization and effective screening protocols. The only founder mutation described in the Mexican population is the deletion of exons 9 to 12 of BRCA1 (BRCA1Δ9-12), and its description focuses on the gene sequence, but no transcription profiles have been generated for individuals who carry this gene. In this study, we describe the transcription profiles of cancer patients and healthy individuals who were heterozygous for PV BRCA1Δ9-12 by analyzing the differential expression of both alleles compared with the homozygous BRCA1 control group using RT-qPCR, and we describe the isoforms produced by the BRCA1 wild-type and BRCA1Δ9-12 alleles using nanopore long-sequencing. Using the Kruskal-Wallis test, our results showed a similar transcript expression of the wild-type allele between the healthy heterozygous group and the homozygous BRCA1 control group. An association between the recurrence and increased expression of both alleles in HBOC patients was also observed. An analysis of the sequences indicated four wild-type isoforms with diagnostic potential for discerning individuals who carry the PV BRCA1Δ9-12 and identifying which of them has developed cancer.

The blood counts of α thalassemia carriers (α-thal) are similar to those of β thalassemia carriers, except for Hemoglobin A2 (Hb A2), which is not elevated. The objective of this study was to determine whether mathematical formulas are effective for detecting suspected α-thal. The data were obtained from the database of the prevention program for detecting couples at risk for having a child with hemoglobinopathy. Red Blood Cells (RBC) indices were analyzed using mathematical formulas, and the sensitivity and negative predictive value (NPV) were calculated. Among 1334 blood counts suspected of α-thal analyzed, only the Shine and Lal and the Support Vector Machine formulas revealed high sensitivity and NPV. Sensitivity was 85.54 and 99.33%, and NPV was 98.93 and 99.93%, respectively. Molecular defects were found in 291, and 81 had normal α genes. Molecular analysis was not performed in 962 of the samples. Based on these results, mathematical formulas incorporating one of these reliable formulas for detecting suspected α or β thalassemia carriers in the program of the automatic analyzers can flag these results, increase the awareness of the primary physicians about the carrier risk, and send an alert with a recommendation for further testing.

Mutations in the SACS gene are associated with autosomal recessive spastic ataxia of Charlevoix-Saguenay disease (ARSACS) or complex clinical phenotypes of Charcot-Marie-Tooth disease (CMT). This study aimed to identify SACS mutations in a Korean CMT cohort with cerebellar ataxia and spasticity by whole exome sequencing (WES). As a result, eight pathogenic SACS mutations in four families were identified as the underlying causes of these complex phenotypes. The prevalence of CMT families with SACS mutations was determined to be 0.3%. All the patients showed sensory, motor, and gait disturbances with increased deep tendon reflexes. Lower limb magnetic resonance imaging (MRI) was performed in four patients and all had fatty replacements. Of note, they all had similar fatty infiltrations between the proximal and distal lower limb muscles, different from the neuromuscular imaging feature in most CMT patients without SACS mutations who had distal dominant fatty involvement. Therefore, these findings were considered a characteristic feature in CMT patients with SACS mutations. Although further studies with more cases are needed, our results highlight lower extremity MRI findings in CMT patients with SACS mutations and broaden the clinical spectrum. We suggest screening for SACS in recessive CMT patients with complex phenotypes of ataxia and spasticity.

OBJECTIVE: To perform molecular diagnosis and pedigree analysis for one case with α-thalassemia who does not conform to the genetic laws, and explore the effects of a newly discovered rare mutation (HBA2:c.*12G>A) on clinical phenotypes.
METHODS: Blood samples of the proband and her family members were collected for blood routine analysis, and the hemoglobin components were analyzed by capillary electrophoresis. The common α- and β-globin gene loci in Chinese population were detected by conventional techniques (Gap-PCR, RDB-PCR). The α-globin gene sequences (HBA1, HBA2) were analyzed by Sanger sequencing.
RESULTS: By analyzing the test results of proband and her family members, the genotype of the proband was -α3.7/HBA2:c.*12G>A, her father was HBA2:c.*12G>A heterozygous mutation carrier.
CONCLUSIONS: This study identifies a rare α-globin gene mutation (HBA2:c.*12G>A) that has not been reported before. It is found that heterozygous mutation carriers present with static α-thalassemia.
UNASSIGNED: HBA2基因非编码区罕见突变分子诊断及家系分析.
UNASSIGNED: 对1例不符合遗传规律的α-地中海贫血病例进行分子诊断及家系分析，探索新发现的罕见突变（HBA2:c.*12G>A）对临床表型的影响。.
UNASSIGNED: 采集先证者及其家系成员的血液样本进行血常规检测，毛细管电泳法进行血红蛋白组分分析，常规技术（Gap-PCR、RDB-PCR）检测中国人群常见的α-及β-珠蛋白基因位点，Sanger测序法分析α-珠蛋白基因序列（HBA1, HBA2）。.
UNASSIGNED: 通过分析先证者及其家系成员的检测结果，检出先证者基因型为-α3.7/HBA2:c.*12G>A，其父亲为罕见α-珠蛋白基因HBA2:c.*12G>A杂合突变携带者。.
UNASSIGNED: 本研究发现了一种未报道的罕见α-珠蛋白基因突变HBA2:c.*12G>A，其杂合突变携带者表现为静止型α-地中海贫血。.

OBJECTIVE: To analyze the clinical phenotype and gene mutation of a genetic coagulation factor XII (FXII) deficiency pedigree and explore the molecular pathogenesis.
METHODS: The activated partial thromboplastin time (APTT) and FXII activity (FXII:C) were detected by clotting method. The FXII antigen (FXII:Ag) was tested with ELISA. All exons and flanks of F12 gene were determined by Sanger sequencing. ClustalX-2.1-win, PROVEAN and Swiss-Pdb Viewer software were used to analyze the conservatism of amino acids at the mutant site, forecast whether the mutant amino acids were harmful and confirm the influence of the mutation on protein structure.
RESULTS: The APTT of the proband prolonged to 71.3 s. The FXII:C and FXII:Ag were decreased to 5% and 6%, respectively. There were two heterozygous missense mutations c.580G>T and c.1681G>A detected in exon 7 and exon 14 of F12 gene, resulting in p.Gly175Cys and p.Gly542Ser, severally. Proband\'s father carried the p.Gly175Cys heterozygous mutation, while mother, brother and daughter had the p.Gly542Ser heterozygous mutation. Software analysis showed that both Gly175 and Gly542 were conserved, the two mutations were harmful and when mutations had occurred, the corresponding sites affected the protein local structure.
CONCLUSIONS: The p.Gly175Cys and p.Gly542Ser compound heterozygous mutations are the molecular pathogenesis of the hereditary coagulation FXII deficiency pedigree. The p.Gly175Cys mutation has been detected for the first time in the world.
UNASSIGNED: F12基因p.Gly175Cys和p.Gly542Ser复合杂合突变导致的遗传性凝血因子Ⅻ缺陷症的家系分析.
UNASSIGNED: 分析1例遗传性凝血因子Ⅻ（FⅫ）缺陷症家系的临床表型和基因突变情况，并探讨其分子致病机制。.
UNASSIGNED: 凝固法检测活化部分凝血活酶时间和FⅫ活性 ；ELISA方法检测FⅫ抗原；Sanger测序法测定F12基因所有外显子及侧翼序列；ClustalX-2.1-win、PROVEAN及Swiss-Pdb Viewer软件分析突变位点氨基酸的保守性、突变氨基酸是否为有害突变及该位点发生突变后对蛋白质结构的影响。.
UNASSIGNED: 先证者活化部分凝血活酶时间延长为71.3 s，FⅫ活性和FⅫ抗原分别降低为5%和6%；其F12基因第7和14外显子分别存在c.580G>T和c.1681G>A杂合错义突变，导致p.Gly175Cys和p.Gly542Ser；先证者父亲携带p.Gly175Cys杂合错义突变；先证者母亲、弟弟和女儿携带p.Gly542Ser杂合错义突变。软件分析结果表明Gly175和Gly542均保守，p.Gly175Cys和p.Gly542Ser为有害突变，突变发生后相应位点会对蛋白质局部结构产生影响。.
UNASSIGNED: p.Gly175Cys和p.Gly542Ser复合杂合突变是先证者家系遗传性FⅫ缺陷症的分子发病机制，其中p.Gly175Cys为国际上首次发现的新突变。.

OBJECTIVE: To identify the genetic mutation of coagulation factor Ⅶ ( F7) gene in a pedigree with coagulation factor Ⅶ (FⅦ) deficiency and explore the molecular pathogenesis.
METHODS: The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), D-dimer (DD), fibrin degradation products (FDP) and coagulation factor Ⅶ activity (FⅦ:C) of the proband and her family members were detected by Sysmex-CS5100 analyzer. All exons and exon-intron boundaries of the F7 gene were amplified by PCR followed by direct sequencing. The detected mutation was confirmed by reverse sequencing. The ClustalW software was used to analyze the conservatism of the mutant site. Pathogenicity of the mutation was assessed with Mutation Taster and PolyPhen-2 online bioinformatics software. Structure of the mutant protein was analyzed using Swiss-PdbViewer software.
RESULTS: The results of routine coagulation tests showed that PT of the proband was markedly extended to 42.5 s, and her FⅦ:C significantly reduced to 2%. The FⅦ:C of her grandmother, mother and sister had slightly reduced to 49%, 51%, and 42%, respectively. These coagulant parameters of her father were within the normal range. Genetic analysis reveled a heterozygous G>A change at cDNA 646 in exon 6 of F7 gene in the proband, resulting in a replacement of glycine at 156 of FⅦ catalytic region with serine (p.Gly156Ser). The sequencing results of other exons and exon-intron boundaries of her F7 gene were normal. The proband\'s grandmother, mother and sister were all the carriers of this missense mutation except her father. Bioinformatics analysis showed that the p.Gly156Ser mutation caused polarity change of the amino acid at this site and formation of side chains, leading to increase of protein instability, which may affect catalytic activity of structural domain. Meanwhile, both Mutation Taster and PolyPhen-2 online bioinformatics software also predicted the pathogenicity of this missense mutation with high scores.
CONCLUSIONS: The heterozygous p.Gly156Ser mutation is the direct cause of the reduced FⅦ in this proband.
UNASSIGNED: 一种新的导致遗传性凝血因子Ⅶ缺陷症的基因突变分析.
UNASSIGNED: 对一例遗传性凝血因子Ⅶ（FⅦ）缺陷症的患者及其家系进行基因分析，探讨其分子发病机制。.
UNASSIGNED: 使用Sysmex-CS5100全自动血凝分析仪检测先证者及其家系成员（共3代8人）的凝血酶原时间（PT）、活化部分凝血活酶时间、凝血酶时间、D-二聚体、纤维蛋白降解产物以及血浆FⅦ的活性（FⅦ:C）水平；PCR法扩增先证者凝血因子Ⅶ基因（ F7）所有外显子和侧翼序列，PCR产物纯化后测序，发现突变位点则反向测序给予证实；使用ClustalW软件对突变位点进行保守性分析；应用Mutation Taster和PolyPhen-2在线生物学软件评估突变氨基酸对FⅦ蛋白结构与功能的危害性；运用Swiss软件对突变建模分析。.
UNASSIGNED: 凝血常规检查结果显示，先证者PT单独性延长至42.5 s；FⅦ:C明显降低，仅为2%；同样先证者外婆、母亲和妹妹的FⅦ:C都有轻度降低，分别为49%、51%和42%；父亲各指标均在正常参考范围。基因分析结果显示，先证者 F7基因第6号外显子cDNA的646位发生G>A杂合突变，导致FⅦ催化区的156位甘氨酸被替换为丝氨酸（p.Gly156Ser）。 F7其他外显子和侧翼序列的测序结果均正常。其外婆、母亲和妹妹均携带c.646G>A杂合突变，父亲为正常野生型。模型构建显示p.Gly156Ser突变使该位点氨基酸极性发生改变并出现侧链，从而使蛋白的不稳定性增加，可能影响所在结构域的催化活性。同时，Mutation Taster和PolyPhen-2两个在线生物信息学软件也高分预测该突变具有致病性。.
UNASSIGNED: 该遗传性凝血因子Ⅶ缺陷症患者FⅦ蛋白p.Gly156Ser错义突变与血浆FⅦ:C水平降低有关。.