Mechanisms of PiT2-loop7 Missense Mutations Induced Pi Dyshomeostasis / 神经科学通报·英文版

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.

Loss-of-function CFTR p.G970D missense mutation might cause congenital bilateral absence of the vas deferens and be associated with impaired spermatogenesis / 亚洲男科学杂志(英文版)

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.

Epididymis cell atlas in a patient with a sex development disorder and a novel NR5A1 gene mutation / 亚洲男科学杂志(英文版)

This study aims to characterize the cell atlas of the epididymis derived from a 46,XY disorders of sex development (DSD) patient with a novel heterozygous mutation of the nuclear receptor subfamily 5 group A member 1 (NR5A1) gene. Next-generation sequencing found a heterozygous c.124C>G mutation in NR5A1 that resulted in a p.Q42E missense mutation in the conserved DNA-binding domain of NR5A1. The patient demonstrated feminization of external genitalia and Tanner stage 1 breast development. The surgical procedure revealed a morphologically normal epididymis and vas deferens but a dysplastic testis. Microfluidic-based single-cell RNA sequencing (scRNA-seq) analysis found that the fibroblast cells were significantly increased (approximately 46.5%), whereas the number of main epididymal epithelial cells (approximately 9.2%), such as principal cells and basal cells, was dramatically decreased. Bioinformatics analysis of cell-cell communications and gene regulatory networks at the single-cell level inferred that epididymal epithelial cell loss and fibroblast occupation are associated with the epithelial-to-mesenchymal transition (EMT) process. The present study provides a cell atlas of the epididymis of a patient with 46,XY DSD and serves as an important resource for understanding the pathophysiology of DSD.

Genetic analysis of two Chinese pedigrees affected with Hereditary hypofibrinemia due to missense variants / 中华医学遗传学杂志

OBJECTIVE@#To retrospectively analyze the clinical phenotypes and genetic variants in two Chinese pedigrees affected with Hereditary hypofibrinemia (IFD) and explore their molecular pathogenesis.@*METHODS@#Two probands and their pedigree members were admitted to the First Affiliated Hospital of Wenzhou Medical University on March 30, 2021 and May 27, 2021, respectively. Clinical phenotypes of the probands were collected, and blood clotting indexes of the probands and their pedigree members were determined. Variants of the FGA, FGB and FGG genes were analyzed by Sanger sequencing, and candidate variants were verified by sequence comparison. Bioinformatic software was used to analyze the conservation of the amino acids and pathogenicity of the proteins. Alteration in protein structure and intermolecular force before and after the variant was analyzed by simulating the protein model.@*RESULTS@#Proband 1, a 18-year-old male, had significantly low plasma fibrinogen activity (Fg:C) and plasma fibrinogen antigen (Fg:Ag), respectively at 0.80 g/L and 1.00 g/L. Proband 2, a 43-year-old male, had slightly low Fg:C and Fg:Ag at 1.35 g/L and 1.30 g/L, respectively. The Fg:C and Fg:Ag of proband 1's father, proband 2's father and son were also below the normal level. Genetic testing showed that proband 1 had harbored a heterozygous missense variant of c.688T>G (p.Phe230Val) in exon 7 of the FGG gene, which was inherited from his father. Proband 2, his father and son all had harbored a heterozygous variant of c.2516A>C (p.Asn839Thr) in exon 6 of the FGA gene. Homology analysis showed that the Phe230 and Asn839 residues were highly conserved among homologous species. Bioinformatic analysis predicted that both p.Phe230Val and p.Asn839Thr were pathogenic variants.@*CONCLUSION@#Analysis of protein simulation model showed that the p.Asn839Thr variant has changed the hydrogen bo`nd between the amino acids, thus affecting the stability of the protein structure. The heterozygous missense variants of p.Phe230Val and p.Asn839Thr probably underlay the IFD in the two pedigrees.

Genetic analysis of a child with Kartagener syndrome due to novel compound heterozygous variants of DNAH5 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical characteristics and genetic basis of a child with Kartagener syndrome (KTS).@*METHODS@#Trio-whole exome sequencing was carried out for the child and his parents, and candidate variants were verified by Sanger sequencing. Changes in protein structure due to missense variants were simulated and analyzed, and the Human Splicing Finder 3.0 (HSF 3.0) online platform was used to predict the effect of the variant of the non-coding region.@*RESULTS@#The child had featured bronchiectasis, sinusitis and visceral inversion. Genetic testing revealed that he has harbored compound heterozygous variants of the DNAH5 gene, namely c.5174T>C and c.7610-3T>G. Sanger sequencing confirmed the existence of the variants. The variants were not found in the dbSNP, 1000 Genomes, ExAC, ClinVar and HGMD databases. Protein structural analysis suggested that the c.5174T>C (p.Leu1725Pro) variant may affect the stability of local structure and its biological activity. The results of HSF 3.0 analysis suggested that the c.7610-3T>G variant has probably destroyed a splicing receptor to affect the transcription process.@*CONCLUSION@#The compound heterozygous variants of the DNAH5 gene probably underlay the pathogenesis in the child. Above finding may facilitate the understanding of the clinical characteristics and genetic basis of KTS, and further expand the spectrum of DNAH5 gene variants.

Analysis of F12 gene variants and molecular mechanisms in patients with coagulation factor Ⅻ deficiency / 中华医学遗传学杂志

OBJECTIVE@#To analyze the sequence of the F12 gene and molecular mechanism for 20 patients with coagulation factor Ⅻ (FⅫ) deficiency.@*METHODS@#The patients were selected from the outpatient department of the Second Hospital of Shanxi Medical University from July 2020 to January 2022. The activity of coagulation factor Ⅷ (FⅧ:C), factor Ⅸ (FⅨ:C), factor Ⅺ (FⅪ:C) and factor Ⅻ (FⅫ:C) were determined by using a one-stage clotting assay. All exons and 5' and 3' UTR of the F12 gene were analyzed by Sanger sequencing to detect the potential variants. Bioinformatic software was used to predict the pathogenicity of the variants, conservation of amino acids, and protein models.@*RESULTS@#The FⅫ:C of the 20 patients has ranged from 0.07% to 20.10%, which was far below the reference values, whilst the other coagulation indexes were all normal. Sanger sequencing has identified genetic variants in 10 patients, including 4 with missense variants [c.820C>T (p.Arg274Cys), c.1561G>A (p.Glu521Lys), c.181T>C (p.Cys61Arg) and c.566.G>C (p.Cys189Ser)], 4 deletional variants c.303_304delCA(p.His101GlnfsX36), 1 insertional variant c.1093_1094insC (p.Lys365GlnfsX69) and 1 nonsense variant c.1763C>A (p.Ser588*). The remaining 10 patients only harbored the 46C/T variant. The heterozygous c.820C>T(p.Arg274Cys) missense variant in patient 1 and the homozygous c.1763C>A (p.Ser588*) nonsense variant in patient 2 were not included in the ClinVar and the Human Gene Mutation Database. Bioinformatic analysis predicted that both variants were pathogenic, and the corresponding amino acids are highly conserved. The protein prediction models suggested that the c.820C>T (p.Arg274Cys) variant may affect the stability of the secondary structure of FⅫ protein by disrupting the original hydrogen bonding force and truncating the side chain, leading to changes in the vital domain. c.1763C>A (p.Ser588*) may produce a truncated C-terminus which may alter the spatial conformation of the protein domain and affect the serine protease cleavage site, resulting in extremely reduced FⅫ:C.@*CONCLUSION@#Among individuals with low low FⅫ:C detected by one-stage clotting assay, 50% have harbored variants of the F12 gene, among which the c.820C>T and c.1763C>A were novel variants underlying the reduced coagulating factor FⅫ.

A recurrent homozygous missense mutation in CCDC103 causes asthenoteratozoospermia due to disorganized dynein arms / 亚洲男科学杂志(英文版)

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.

A child with diffuse mesangial sclerosis caused by a missense mutation of TRPC6 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic etiology and clinical outcome of a child with steroid-resistant nephrotic syndrome and diffuse mesangial sclerosis.@*METHODS@#Genomic DNA was extracted from peripheral blood leukocytes of the proband and his parents. Targeted capture - next generation sequencing and Sanger sequencing were carried out. Candidate variant was verified by segregation analysis in his family.@*RESULTS@#A heterozygous missense variant of the TRPC6 gene, namely c.325G>A (p.Gly109Ser), was detected in the proband. The same variant was not detected in either parent. According to the guidelines for the interpretation of sequence variants developed by American College of Medical Genetics and Genomics, the variant was predicted as pathogenic.@*CONCLUSION@#The missense variant of the TRPC6 gene probably underlay the diffuse mesangial sclerosis in this patient. Above finding has expanded the phenotypic spectrum of the TRPC6 gene.

Clinical and laboratory features compared between JAK2 exon12 and JAK2 V617F mutated polycythemia vera / 中华血液学杂志

Objective: To compare clinical and laboratory features between JAK2 exon12 and JAK2 V617F mutated polycythemia vera (PV) . Method: We collected data from 570 consecutive newly-diagnosed subjects with PV and JAK2 mutation, and compared clinical and laboratory features between patients with JAK2 exon12 and JAK2 V617F mutation. Results: 543 (95.3%) subjects harboured JAK2 V617F mutation (JAK2 V617F cohort) , 24 (4.2%) harboured JAK2 exon12 mutations (JAK2 exon12 cohort) , and 3 (0.5%) harboured JAK2 exon12 and JAK2 V617F mutations. The mutations in JAK2 exon12 including deletion (n=10, 37.0%) , deletion accompanied insertion (n=10, 37.0%) , and missense mutations (n=7, 25.9%) . Comparing with JAK2 V617F cohort, subjects in JAK2 exon12 cohort were younger [median age 50 (20-73) years versus 59 (25-91) years, P=0.040], had higher RBC counts [8.19 (5.88-10.94) ×10(12)/L versus 7.14 (4.11-10.64) ×10(12)/L, P<0.001] and hematocrit [64.1% (53.7-79.0%) versus 59.6% (47.2%-77.1%) , P=0.001], but lower WBC counts [8.29 (3.2-18.99) ×10(9)/L versus 12.91 (3.24-38.3) ×10(9)/L, P<0.001], platelet counts [313 (83-1433) ×10(9)/L versus 470 (61-2169) ×10(9)/L, P<0.001] and epoetin [0.70 (0.06-3.27) versus 1.14 (0.01-10.16) IU/L, P=0.002] levels. We reviewed bone marrow histology at diagnosis in 20 subjects with each type of mutation matched for age and sex. Subjects with JAK2 exon12 mutations had fewer loose megakaryocyte cluster (40% versus 80%, P=0.022) compared with subjects with JAK2 V617F. The median follow-ups were 30 months (range 4-83) and 37 months (range 1-84) for cohorts with JAK2 V617F and JAK2 exon12, respectively. There was no difference in overall survival (P=0.422) and thrombosis-free survival (P=0.900) . Conclusions: Compared with patients with JAK2 V617F mutation, patients with JAK2 exon12 mutation were younger, and had more obvious erythrocytosis and less loose cluster of megakaryocytes.

Clinical phenotype and gene mutation analysis of 12 patients with hereditary protein C deficiency in different families / 中华血液学杂志

Objective: To investigate the molecular pathogenesis and clinical features of unrelated 12 patients with inherited coagulation protein C (PC) deficiency in Chinese population. Methods: The PC activity (PC:A) and PC antigen (PC:Ag) were detected by chromogenic substrate and enzyme linked immunosorbent assay, respectively. The nine exons and flanking sequences of the protein C (PROC) gene were amplified by polymerase chain reaction with direct sequencing, and the suspected mutations were validated by reverse sequencing (clone sequencing for deletion mutations) . Results: The PC:A of the 12 probands decreased significantly, ranging from 18% to 55%, and the PC:Ag of the 10 probands decreased significantly. Eleven mutations were found, out of which four mutations [c.383G>A (p.Gly128Asp) , c.997G>A (p.Ala291Thr) , c.1318C>T (p.Arg398Cys) , and c.532G>C (p.Leu278Pro) ] were discovered for the first time. Six mutations were in the serine protease domain, four mutations were located in epidermal growth factor (EGF) -like domains, and one mutation was located in activation peptide. There were two deletion mutations (p.Met364Trp fsX15 and p.Lys192del) , and the rest were missense mutations. Mutations p.Phe181Val and p.Arg189Trp were identified in three unrelated families. All mutations may be inherited, and consanguineous marriages were reported in two families. Among the probands, nine cases had venous thrombosis, two cases had poor pregnancy manifestations, and one case had purpura. Conclusion: Patients with PC deficiency caused by PROC gene defects are prone to venous thrombosis, especially when there are other thrombotic factors present at the same time.

Performance of mutation pathogenicity prediction tools on missense variants associated with 46, XY differences of sex development

OBJECTIVES: Single nucleotide variants (SNVs) are the most common type of genetic variation among humans. High-throughput sequencing methods have recently characterized millions of SNVs in several thousand individuals from various populations, most of which are benign polymorphisms. Identifying rare disease-causing SNVs remains challenging, and often requires functional in vitro studies. Prioritizing the most likely pathogenic SNVs is of utmost importance, and several computational methods have been developed for this purpose. However, these methods are based on different assumptions, and often produce discordant results. The aim of the present study was to evaluate the performance of 11 widely used pathogenicity prediction tools, which are freely available for identifying known pathogenic SNVs: Fathmn, Mutation Assessor, Protein Analysis Through Evolutionary Relationships (Phanter), Sorting Intolerant From Tolerant (SIFT), Mutation Taster, Polymorphism Phenotyping v2 (Polyphen-2), Align Grantham Variation Grantham Deviation (Align-GVGD), CAAD, Provean, SNPs&GO, and MutPred. METHODS: We analyzed 40 functionally proven pathogenic SNVs in four different genes associated with differences in sex development (DSD): 17β-hydroxysteroid dehydrogenase 3 (HSD17B3), steroidogenic factor 1 (NR5A1), androgen receptor (AR), and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF>0.01) benign SNVs found in the same four DSD genes. The quality of the predictions was analyzed using six parameters: accuracy, precision, negative predictive value (NPV), sensitivity, specificity, and Matthews correlation coefficient (MCC). Overall performance was assessed using a receiver operating characteristic (ROC) curve. RESULTS: Our study found that none of the tools were 100% precise in identifying pathogenic SNVs. The highest specificity, precision, and accuracy were observed for Mutation Assessor, MutPred, SNP, and GO. They also presented the best statistical results based on the ROC curve statistical analysis. Of the 11 tools evaluated, 6 (Mutation Assessor, Phanter, SIFT, Mutation Taster, Polyphen-2, and CAAD) exhibited sensitivity >0.90, but they exhibited lower specificity (0.42-0.67). Performance, based on MCC, ranged from poor (Fathmn=0.04) to reasonably good (MutPred=0.66). CONCLUSION: Computational algorithms are important tools for SNV analysis, but their correlation with functional studies not consistent. In the present analysis, the best performing tools (based on accuracy, precision, and specificity) were Mutation Assessor, MutPred, and SNPs&GO, which presented the best concordance with functional studies.

Identification of two novel variants of the PCCB gene in a pedigree affected with propionic acidemia / 中华医学遗传学杂志

OBJECTIVE@#To detect pathogenic variants in a pedigree affected with propionic acidemia (PA).@*METHODS@#The proband was subjected to high-throughput next-generation sequencing. Suspected variants were validated by Sanger sequencing of his family members. mRNA was extracted from peripheral blood lymphocytes from the proband's father in order to verify the impact of the splicing variant by RT-PCR combined with Sanger sequencing. The pathogenicity of the missense variant was predicted by using PolyPhen-2, Mutation Taster, SIFT, COBALT and HOPE software.@*RESULTS@#The proband was found to harbor compound heterozygous variants of the PCCB gene, namely c.184-2A>G and c.733G>A (p.G245S), which were respectively inherited from his father and mother. RT-PCR combined with Sanger sequencing confirmed skipping of exon 2 during transcription. Bioinformatic analysis indicated the c.733G>A (p.G245S) variant to be damaging.@*CONCLUSION@#The two variants of the PCCB gene probably underlay the disease in this patient. Above findings have enriched the spectrum of PCCB gene variants.

Genetic analysis of a child with co-commitment progressive multifocal leukoencephalopathy and X-linked hyper IgM syndrome / 中华医学遗传学杂志

OBJECTIVE@#To detect variant of the CD40L gene and infection of Jamestown Canyon virus (JCV) in a 7-year-and-9-month-old boy with co-commitment progressive multifocal leukoencephalopathy (PML) and X-linked hyper IgM syndrome (XHIGM).@*METHODS@#Peripheral blood samples of the child and his parents were collected for the extraction of genomic DNA. The 5 exons and exon/intronic boundaries of the CD40L gene were subjected to PCR amplification and sequencing. Suspected variants were analyzed by using bioinformatic software. The JCV gene was amplified from genomic DNA by nested PCR and sequenced.@*RESULTS@#The child was found to harbor a hemizygous c.506 A>C (p.Y169S) missense variant in exon 5 of the CD40L gene. The variant may affect the TNFH domain of the CD40L protein and result in structural instability and loss of hydrophobic interaction between CD40L and CD40. As predicted by PolyPhen2 and SIFT software, the variant was probably damaging (score = 1.00) and deleterious (score= -8.868). His mother was found to be a heterozygous carrier, while the same variant was not found in his father. Gel electrophoresis of the nested PCR product revealed presence of target JCV band, which was confirmed to be 99% identical with the JCV gene by sequencing.@*CONCLUSION@#The patient was diagnosed with co-commitment XHIGM and PML based on the testing of the CD40L gene and JCV infection.

Analysis of MCCC2 gene variant in a pedigree affected with 3-methylcrotonyl coenzyme A carboxylase deficiency / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a child with clinically suspected 3-methylcrotonyl-coenzyme A carboxylase deficiency (MCCD).@*METHODS@#Genomic DNA was extracted from peripheral blood samples of the proband and her parents. Whole exome sequencing was used to screen pathogenic variant in the proband. Suspected variant was verified by Sanger sequencing. Impact of the variant on the structure and function of protein product was analyzed by using bioinformatic software.@*RESULTS@#Sanger sequencing showed that the proband has carried homozygous missense c.1342G>A (p.Gly448Ala) variant of the MCCC2 gene, for which her mother was a heterozygous carrier. The same variant was not detected in her father. The variant was predicted to be pathogenic by PolyPhen-2 and Mutation Taster software, and the site was highly conserved among various species. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.1342G>A (p.Gly448Ala) variant of MCCC2 gene was predicted to be likely pathogenic(PM2+PP2-PP5).@*CONCLUSION@#The homozygous missense variant of the MCCC2 gene c.1342G>A (p.Gly448Ala) probably underlay the molecular pathogenesis of the proband. Genetic testing has confirmed the clinical diagnosis.

Identification of pathogenic variant in a Chinese pedigree affected with non-syndromic cleft lip and palate / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a Chinese pedigree affected with non-syndromic cleft lip and cleft palate (NSCLP).@*METHODS@#With informed consent obtained, members of the pedigree were subjected to clinical examination and history taking to exclude syndromic cleft lip and palate. One affected member was subjected to whole-exome sequencing and bioinformatics analysis. Candidate variant was verified by Sanger sequencing and co-segregation analysis of her family members and 100 unrelated healthy individuals.@*RESULTS@#Whole-exome sequencing and co-segregation analysis showed that all affected members of this pedigree have carried a heterozygous missense c.253A>G (p.Cys85Arg) variant in exon 4 of the IRF6 gene, which has co-segregated with the phenotype and was not found among the 100 unrelated healthy individuals.@*CONCLUSION@#The missense c.253A>G variant in exon 4 of the IRF6 gene probably underlay the NSCLP in this pedigree.

Serological and molecular study of a novel B(A) allele with multiple missense mutations / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for an individual suspected as AwB subtype through DNA sequencing.@*METHODS@#ABO serology was carried out with the standard tube method. To identify the ABO gene haplotype, the amplicons of exon 7 were cloned and sequenced.@*RESULTS@#Serological results showed that the forward typing was AwB and the reverse typing was B. Sequencing analysis revealed that the sample has contained an O01 allele in addition with c.297A>G, c.657C>T, c.796C>A, c.803G>C, c.930G>A variants as compared with the A101 allele.@*CONCLUSION@#Through sequencing analysis, the sample with an AwB subtype by serological testing was identified as a novel B(A) phenotype, which was unreported previously.

17 beta-hydroxysteroid dehydrogenase 3 deficiency due to novel compound heterozygous variants of HSD17B3 gene in a sib pair / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a sib pair featuring 17beta-hydroxysteroid dehydrogenase type 3 deficiency.@*METHODS@#Genomic DNA was extracted from the proband, her sister, and their parents, and was subjected to sequencing analysis with a gene panel for sexual development. Suspected variant was verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#Both the proband and her sister were found to harbor novel compound heterozygous missense variants of the HSD17B3 gene, namely c.839T>C (p.Leu280Pro) and c.239G>T (p.Arg80Leu), which were derived respectively from their mother and father. The variants were unreported previously and predicted to be deleterious by PolyPhen2, MutationTaster and other online software. Based on the American College of Medical Genetics and Genomics standards and guidelines, both c.839T>C(p.Leu280Pro) and c.239G>T (p.Arg80Leu) were predicted to be likely pathogenic (PM2+PP1+PP2+PP3+PP4, PM2+PM5+PP1+PP2+PP3+PP4).@*CONCLUSION@#The compound heterogeneous variants of the HSD17B3 gene probably underlay the disease in this sib pair. 17beta-hydroxysteroid dehydrogenase type 3 deficiency may lack specific clinical features and laboratory index, genetic testing can facilitate a definitive diagnosis.

A study on KIF1A gene missense variant analysis and its protein expression and structure profiles of an autism spectrum disorder family trio / 中华医学遗传学杂志

OBJECTIVE@#To analyze the pathogenic variants of the KIF1A gene and its corresponding protein structure in an autism spectrum disorder (ASD) family trio carrying harmful missense variants in the KIF1A gene.@*METHODS@#The peripheral blood DNA of the patient and his parents was extracted and sequenced using whole exome sequencing (WES) technology and verified by Sanger sequencing. Bioinformatics software SIFT, PolyPhen-2, Mutation Taster, and CADD software were used to analyze the harmfulness and conservation of variants. The Human Brain Transcriptome (HBT) database was used to analyze the expression of the KIF1A gene in the brain. PredictProtein and SWISS-MODEL were further used to predict the secondary structure and tertiary structure of KIF1A wild-type protein and variant protein. PyMOL V2.4 was utilized to investigate the change of hydrogen bond connection after protein variant.@*RESULTS@#The WES sequencing revealed a missense variant c.664A>C (p.Asn222His) in the child's KIF1A gene, and this variant was a de novo variant. The harmfulness prediction results suggest that this variant is harmful. By analyzing expression level of KIF1A gene in the brain. It is found that KIF1A gene widely expressed in various brain regions during embryonic development. By analyzing the variant protein structure, the missense variant of KIF1A will cause many changes in the secondary structure of protein, such as alpha-helix, beta-strand, and protein binding domain. The connection of hydrogen bond and spatial structure will also change, thereby changing the original biological function.@*CONCLUSION@#The KIF1A gene may be a risk gene for ASD.

Different Subtypes Caused by c.721C>T Substitution in the Exon 7 of ABO Gene / 中国实验血液学杂志

OBJECTIVE@#To analyze the different subtypes caused by c.721C>T substitution in the exon 7 of the ABO gene, and to investigate the related molecular mechanism of different antigens expression.@*METHODS@#ABO subtypes in 7 samples were identified by standard serological methods. The exons 6, 7, and adjacent intron of ABO gene were amplified by Polymerase Chain Reaction (PCR), and the PCR products were analyzed by direct DNA sequencing and cloning sequencing.@*RESULTS@#ABO subtypes phenotypes were A@*CONCLUSION@#c.721C>T substitution in the ABO gene causes p.Arg241Trp exchange resulting in the decreasing of GTA or GTB activities and weaker antigen expression. O.01.07 is a null allele which cannot form a functional catalytic enzyme has no effect on A

Analysis of SIK3 gene variation in a boy with autism spectrum disorder complicated with epilepsy / 中华医学遗传学杂志

OBJECTIVE@#To study the genetic variants of a child with Autism Spectrum Disorder (ASD) combined with epilepsy, and explore its possible pathogenic mechanism.@*METHODS@#Clinical data of the child were collected and evaluated, whole-exome sequencing (WES) technology was used to explore the genetic variants sites of the child and his parents and candidate genes were filtered out. Sanger sequencing were performed to verify the variants identified by WES and PolyPhen2 was utilized to predict the function of these variants. qPCR was carry out to determine the expression of the variant gene.@*RESULTS@#The proband carried a compound heterozygous mutation in the SIK3 gene (Chr11 q23.3, NM_025164.6), which contains a missense mutation c.1295A>G (p.N432S) inherited from the father and a deletion [c.2389_2391del(p.797del)] inherited from the mother. Both mutation sites are highly conservative, and PolyPhen2 predicted (c.1295A>G [p.N432S]) to be harmful. Compared to the mother, expression of SIK3in mRNA level in the peripheral blood of the proband and his father were both significantly decreased; compared to normal child, SIK3 expression in the peripheral blood of the proband and two other children with ASD were all decreased significantly too. In addition, studies on mice found that Sik3 gene has a marked higher level of expression in the brain.@*CONCLUSION@#The SIK3 gene variants may probably be associated with ASD. The detailed mechanism needs to be studied further, which may involve lipid metabolism dysfunction in the brain.