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To explore the genetic etiology of a child featuring recurrent oral ulcer.Clinical data of the child was collected. Whole exome sequencing was carried out for her. Candidate variant was verified by low-coverage massive parallel copy number variation sequencing (CNV-seq) of the family trio.The child, a 6-year-old girl, has featured recurrent fever and ulcers of the oral mucosa, vulvar and perianal regions. No pathogenic variant was found by whole exome sequencing. However, analysis of chromosome copy number variation using the whole exome sequencing data has revealed mosaicism of trisomy 8. CNV-seq assay has verified the variant in the child, with the percentage of mosaicism being 73%. No abnormality was found in neither of her parents.A case of mosaicism trisomy 8 with recurrent oral ulcer as the first symptom was diagnosed, which has enriched the phenotypic data of trisomy 8 syndrome.

Oligohydramnios or polyhydramnios, is associated with chromosomal aberrations, particularly aneuploidy. However, its correlation with copy number variation (CNV) remains unclear.We retrospectively analyzed 428 cases with an abnormal level of amniotic fluid, comprising of 139 cases of single ultrasound findings (SU group) and 289 cases of multiple ultrasound findings (MU group), by CNV sequencing (CNV-Seq) and followed their pregnancy outcomes.The overall detection rate of clinically significant findings was 8%, with 5% in the SU group and 11% in MU group. Besides, 18 microdeletion/microduplication syndromes were detected, with the highest rate of renal cysts and diabetes syndrome (22%, 4/18). Also, the rate of termination of pregnancy in MU group was much higher than that in the SU group (29% vs. 10%, ***p 0.001), and in the MU-oligohydramnios subgroup, it was the highest (34%), regardless of cases with chromosomal anomaly and lost to follow-up.Our results showed that the abnormal level of amniotic fluid, especially combined with other ultrasound abnormalities, is closely related to chromosomal abnormalities and genetic CNVs. CNV-Seq may be useful in investigating pregnancies with an abnormal amniotic fluid level.

To summarize the genetic diagnosis, low-depth copy number variation sequencing (CNV-seq) and prenatal finding in 7 fetuses with 2p16.3 deletions only involving the NRXN1 gene.The 7 fetuses have all been found to have loss of heterozygosity at 2p16.3 by CNV-seq, which were verified by quantitative real-time PCR (qPCR). Specific regions of NRXN1 gene deletions were identified, and the CNVs were verified in their parents. Outcome of the pregnancies were followed up.Among 16 502 prenatal samples, 7 fetuses were found to harbor a 120 kb ~ 900 kb microdeletion in the 2p16.3 region, which yielded a prevalence of 0.424‰. The deleted region mainly involved 50 200 000-51 880 000 positions of chromosome 2 and involved only the NRXN1 gene. All of the 7 fetal CNVs were confirmed by qPCR, including 2 cases with heterozygous deletion of exons 1 to 6, 1 with heterozygous deletion of exons 1 to 19, 1 with heterozygous deletion of exons 19 to 22, and 3 with heterozygous deletion of introns 6 to 7 of the NRXN1 gene. Verification in the parents had found that one deletion was inherited from the father, 1 was from the mother, 2 cases were de novo in origin, whilst the remaining 3 had refused parental verification. After genetic counseling, one couple had elected induced abortion, 1 case has not been born yet, whilst the other 5 cases were born healthy. Follow up had identified no mental abnormalities among the children.Seven fetuses with heterozygous 2p16.3 deletions only involving the NRXN1 gene were detected by CNV-seq. The specific deletion of the NRXN1 gene was verified by qPCR. Prenatal genetic counseling and fertility guidance has been provided to the particular family by combining the results of CNV testing, pedigree analysis and pregnancy outcome.

To explore the clinical features and genetic basis for a child with Bainbridge-Ropers syndrome (BRPS).Clinical data of the child were retrospectively analyzed. Copy number variation sequencing (CNV-seq) and trio based whole exome sequencing (trio-WES) were carried out. Prenatal diagnosis was provided for a at risk fetus from the pedigree, and genotype phenotype correlation was summarized through a literature review.The proband, a 6-year-old boy, has presented with feeding difficulties, specific craniofacial features, global developmental delay and intellectual disability, which has not improved after rehabilitation treatment. CNV-seq analysis of the patient showed no obvious abnormalities. A de novo heterozygous truncating variation, c.1448dupT (p.T484Nfs*5), was identified in the ASXL3 gene by trio-WES, which was a previously reported pathogenic variant. So far 14 Chinese patients with BRPS and ASXL3 variants have been reported. All patients have shown specific craniofacial features and delayed motor and speech development, and harbored 12 loss of function ASXL3 variants, which were de novo in origin and have clustered in exons 11 and 12 of the ASXL3 gene.The heterozygous frameshift c.1448dupT (p.T484Nfs*5) variant of the ASXL3 gene probably underlay the disorder in this patient. BRPS should be considered in infants with feeding difficulties, special craniofacial features, global developmental delay and hand anomalies, and WES can help to delineate the pathogenesis and establish the definite diagnosis.

To assess the diagnostic value of copy number variation sequencing (CNV-seq) in the genetic etiology of fetuses with nasal bone dysplasia (NBD).A total of 217 fetuses discovered with NBD from December 2017 to December 2020 were divided into the isolated NBD group and NBD combined with other anomalies group, for which copy number variations (CNVs) were analyzed.A total of 40 fetal abnormalities were detected in 217 cases, with an overall abnormal rate of 18.4%. These included 31 cases with aneuploidies (14.3%, 31/217) and 9 cases with genomic CNVs (4.1%, 9/217). Five cases of trisomy 21 (3.5%, 5/144) and two CNVs cases with unknown clinical significance (1.4%, 2/144) were detected in the isolated group. As for the combined NBD group, 26 aneuploidies (35.6%, 26/73), including 19 cases with trisomy 21, 6 cases with trisomy 18, 1 case with trisomy 13, 5 cases with pathogenic CNVs (6.8%, 5/73), and 2 cases with CNVs of unknown clinical significance (2.7%, 2/73) were detected. A significant difference was detected between the two groups (P0.01).The detection rate of CNV-seq is high for chromosomal aneuploidies and pathogenic CNVs in fetuses with NBD, particularly in those combined with other ultrasonic abnormalities.

To assess the value of low-depth whole-genome copy number variation sequencing (CNV-seq) for the analysis of chromosomal copy number variations among fetuses with echogenic bowel (EB).A total of 163 fetuses were included in this study. Amniotic fluid (162 cases) or chorionic villi (1 case) were collected and subjected to CNV-seq for the analysis of CNVs.Thirteen (8.0%) pathogenic CNVs were detected, including 9 (5.5%) aneuploidies and 4 (2.4%) CNVs. The detection rate of the isolated EB group and combined EB group were 1.7% (1/58) and 11.4% (12/105), respectively. There was a significant difference between the two groups (P0.05). A Xp22.1 duplication was detected in both groups, and the fetuses were predicted as female DMD carriers and born healthy. Nine cases of aneuploidies and 2 (likely) pathogenic CNVs were identified in the combined EB group, all of them have warranted induced labor.The prevalence of chromosomal aneuploidies and pathogenic CNVs in fetuses with combined EB was much higher than isolated EB, and most of them may warrant termination of pregnancy. Compared with isolated EB, more attention should be paid to combined EB, for which prenatal diagnosis and genetic counseling should be carried out in time.

Objectives To evaluate the clinical potential of a higher resolution noninvasive prenatal screening (NIPS-Plus) test for detection of microdeletion/microduplication syndromes (MMS) in addition to common aneuploidies. Methods In a multicenter prospective study, 37,002 pregnant women with unremarkable first-trimester ultrasound scans had a NIPS-Plus test. Ultrasound screen positive women were not included in this study. Results Of 36,970 ultrasound negative women there were 291 NIPS-Plus screen positive results indicating 237 aneuploidies and 54 MMS. Following amniocentesis, 171 (72%) were confirmed as genuine, comprising 3 T13s, 10 T18s, 61 T21s, 70 SCAs and 27 MMS. The PPV for MMS with unremarkable ultrasound findings was 50%. Routine clinical examination of children born from NIPS-Plus negative pregnancies revealed no obvious signs of chromosome disease syndromes at one year of age. Conclusions NIPS-Plus has the potential for clinical utility not only for routine aneuploid screening but also for MMS that do not show overt signs during early pregnancy ultrasound screening. We suggest that ultrasound with NIPS-Plus in combination with appropriate counselling could be considered as a comprehensive first-tier prenatal screening approach for all pregnant women.

To explore the genetic basis for a girl with distinctive facial features, epilepsy, intellectual disability, chronic constipation and hypopigmentation of neck and upper extremities.Whole exome sequencing was carried out for the proband. Candidate variant was verified by Sanger sequencing.The proband was found to harbor a heterozygous nonsense c.586GT (p.Glu196*) variant of the ZEB2 gene, which was unreported previously. The variant was not detected in either parent.The ZEB2 gene c.586GT (p.Glu196*) variant probably underlay the Mowat-Wilson syndrome in this patient. Hypopigmentation in the neck and upper extremities may be related to Mowat-Wilson syndrome. Prenatal diagnosis was recommended for subsequent pregnancies.

Neurodevelopmental disorders (NDDs) are a genetically heterogeneous group of diseases, affecting 1%-3% of children. Whole-exome sequencing (WES) has been widely used as a first-tier tool for identifying genetic causes of rare diseases. Trio-WES was performed in a cohort of 74 pedigrees with NDDs. Exome-based copy number variant (CNV) calling was incorporated into the traditional single-nucleotide variant (SNV) and small insertion/deletion (Indel) analysis pipeline for WES data. An overall positive diagnostic yield of 54.05% (40/74) was obtained in the pipeline of combinational SNV/Indel and CNV analysis, including 35.13% (26/74) from SNV/Indel analysis and 18.92% (14/74) from exome-based CNV analysis, respectively. In total, SNV/Indel analysis identified 38 variants in 28 different genes, of which 24 variants were novel; exome-based CNV analysis identified 14 CNVs, including 2 duplications and 12 deletions, which ranged from 440 bp (single exon) to 16.86 Mb (large fragment) in size. In particular, a hemizygous deletion of exon 1 in the SLC16A2 gene was detected. Based on the diagnostic results, two families underwent prenatal diagnosis and had unaffected babies. The incorporation of exome-based CNV detection into conventional SNV/Indel analysis for a single trio-WES test significantly improved the diagnostic rate, making WES a more powerful, practical, and cost-effective tool in the clinical diagnosis of NDDs.

Aim Families with an adverse history of monogenic disease focus on single‐gene diagnosis instead of low‐depth whole‐genome sequence, during subsequent pregnancies. The aim of this study was to assess the potential usefulness of low‐depth whole‐genome sequencing (copy number variant sequencing [CNV‐seq]) detection following monogenic disease exclusion in prenatal diagnosis. Methods A total of 285 families with a history of monogenic disease (of 41 different types; eliminated during the current pregnancy) were recruited and retrospectively analyzed. Low‐depth whole‐genome sequencing (CNV‐Seq, Next‐Seq CN500 platform) was performed for all fetuses. Results The CNV detection results of the 285 samples were as follows: one case of 18‐trisomy chimera (0.35%), one case of pathogenic 3q29 microdeletion syndrome CNV (0.35%), four cases of variant of uncertain significance (VUS) CNVs (1.40%), and four cases of Duchenne muscular dystrophy (DMD) carriers (1.40%); and the remaining samples were normal (96.15%). Of note, 2/285 (0.70%) samples still exhibited pathogenic abnormalities. All positive samples were followed up where the two cases of pathogenic abnormalities elected the pregnancy termination, while the four VUS cases and four DMD‐carrier cases were born healthy. Conclusion In cases where prenatal fetal monogenic disease has been ruled out, CNV detection is still beneficial and should be performed to prevent missed pathogenic CNVs. However, the costs need to be balanced against benefits, and the research will need to assess other types of testing.

To assess the value of copy number variation sequencing (CNV-seq) and karyotyping in the prenatal diagnosis for carriers of balanced translocations.Clinical records of 135 amniocentesis samples of balanced translocation carriers undergoing simultaneous CNV-seq and karyotyping were analyzed. Chromosomal aberrations were defined as those can definitely lead to birth defects definitely, which included chromosomal numerical abnormality, large deletion/duplication and pathogenic copy number variations (pCNVs).The detection rates for karyotyping and CNV-seq were 4.44% (6/135) and 5.93% (8/135) respectively, and the latter had a detection rate of 1.48(2/135) higher than the former. A total of 68 fetal chromosomal translocations were detected by karyotying analysis.For couples carrying a balanced translocation, simultaneous CNV-seq and karyotyping is conducive to the detection of fetal chromosomal abnormalities and genetic counseling.

To analyze the clinical symptom and parental origin of patients with MECP2 duplication syndrome in order to provide a basis for genetic counseling and prenatal diagnosis.Clinical symptoms of four patients who were diagnosed with MECP2 duplication syndrome by copy number variation sequencing (CNV-Seq) were reviewed. The maternal origin of the duplications were verified.All patients were males, and CNV-Seq revealed that they have all harbored a duplication in the Xq28 region spanning 0.32 ~ 0.86 Mb, which were derived from asymptomatic mothers. The clinical symptoms of three patients with three copies included delayed speech, intellectual disability, and muscular hypotonia, while the patient with four copies had died at 6 months after birth, with clinical symptoms including recurrent infections, seizures, and spasticity.The four cases of MECP2 duplication syndrome have shown complete penetrance and have all derived from asymptomatic mothers. As a stable and reliable method, CNV-Seq can accurately detect the MECP2 duplication syndrome.

Duchenne muscular dystrophy (DMD), one of the most common progressive and severely disabling neuromuscular diseases in children, can be largely attributed to the loss of function of the DMD gene on chromosome Xp21.2-p21.1. This paper describes the case of a 10-year-old boy diagnosed with DMD. Whole exome sequencing confirmed the hypothesized large partial exonic deletion of c.7310-11543_7359del (chrX:g.31792260_31803852del) spanning exon 51 and intron 50 in DMD. This large deletion was verified to be de novo by PCR, and the two breakpoints were further confirmed by Sanger sequencing and long-read whole-genome sequencing. Notably, this partial exonic deletion was the only complex variation in the deep intron regions or intron–exon junction regions in DMD. In addition, the case study demonstrates the clinical importance of using multiple molecular genetic testing methods for the diagnosis of rare diseases.

To identify the pathogenic variants of 4 patients with hemolytic anemia of unknown cause.Peripheral blood samples of the patients and their family members were collected to extract DNA. The coding region and splice region in all exons of gene of erythrocyte related diseases were analyzed by using target sequence capture and high-throughput sequencing technology. Suspected pathogenic variants were verified by PCR combined Sanger sequencing technology.Each of the probands was detected two compound heterozygous variants, and CDA II was diagnosed. Six variants were detected in the 4 probands, four variants were reported and the other two were first reported.By high-throughput sequencing, gene variant of CDA II be analyzed fast and accurately. It is an effective supplement to convenional diagnostic methods. Furthermore, the novel variant sites have enriched the variant database of the SEC23B gene.

To analyze the prenatal diagnosis, parental verification and pregnancy outcome of 6 fetuses with 22q11.2 microdeletion syndrome.Copy number variation sequencing (CNV-seq)and chromosomal microarray analysis (CMA) were carried out for the fetuses.The fetuses were found to harbor 2.54-3.2 Mb microdeletions of the 22q11.2 region, among which one was maternally inherited and one was paternally inherited. Two parents opted to continue with the pregnancy, and 4 chose induced labor. One fetus was found to have tetralogy of Fallot, while two carrier parents and one fetus appeared to have normal phenotype.22q11.2 microdeletions identified upon prenatal diagnosis should be treated carefully, with ultrasonic scan and parental verification taken into account.

To explore the genetic basis for a neonate with Pierre-Robin sequence.The child was subjected to chromosomal karyotyping, single nucleotide polymorphism array (SNP-array)-based comparative genomic hybridization and fluorescence in situ hybridization (FISH) analysis.The child has featured microgthnia, glossoptosis, upper airway obstruction, mandible dehiscence and short neck. He was found to have a karyotype of 46,XY,der(4)add(4)(q34). Her mother's karyotype was determined as 46,XX,t(1;4)(q43;q34), while his father was 46,XY. SNP-array analysis suggested the child to be arr [hg19] 1q42.2q44 (232 527 958-249 202 755)× 3; 4q34.3q35.2 (168 236 901-190 880 409)× 1. The result of SNP-array for both parents was normal. FISH analysis confirmed that his mother has carried a balanced t(1;4)(q42;34) translocation. The aberrant chromosome 4 in the child has derived from his mother's translocation, which gave rise to partial 1q trisomy and 4q monosomy.The 1q42.2q44 duplication and 4q34.3q35.2 deletion of the child probably underlay his abnormal phenotype of Pierre-Robin sequence.

BackgroundDistal 8p duplication is rare but clinically significant. Duplication syndrome results in variable phenotypes, such as developmental delay, intellectual disability, and malformation of the heart. We aimed to provide a better understanding of the phenotypes by studying duplication and its effects in a single family.MethodsIn a family with a previously induced labor (second fetus) at 12 weeks gestation due to increased nuchal translucency (3.5 mm), copy number variation sequencing (CNV-seq) revealed a 16.22 Mb deletion of 8p23.3p22. For their subsequent pregnancy, the family requested a prenatal diagnosis as well as CNV-seq, karyotyping and FISH testing of all family members.ResultsThe first and third children were found to have a 16.22 Mb duplication of 8p23.3p22, containing the 8p23.1 duplication syndrome region. The duplication was inherited from their father, a carrier with a translocation of 8p22 and 22q13. We confirmed that the duplication site was located on chromosome 22q13 by combining the results of CNV-seq, karyotype and FISH. The first child is a 7.5-year-old boy. At one month old, he was diagnosed with a ventricular septal defect and treated surgically at age four. His growth and intelligence developed well, and he performed well in school. His primary issue is an inability to distinguish between the blade alveolars and retroflexes in speech. The third fetus had a normal ultrasound index from beginning until birth. The family elected to continue the pregnancy, and the baby was born healthy, providing us the opportunity to evaluate the effects of 8p23.3p22 duplication by comparison with the brother.ConclusionOur study makes a significant contribution to the literature because this relatively rare condition can have significant phenotypical consequences, and an understanding of the inheritance and variability of phenotypes caused by this mutation is essential to an increased understanding of the condition.

To analyze the clinical phenotype and genetic variants in five Chinese pedigrees affected with Dysferlinopathy.Next generation sequencing (NGS) was carried out for the probands from the five pedigrees. Suspected variants were validated by Sanger sequencing. Pathogenicity of the variants was assessed based on the standards and guidelines by the American College of Medical Genetics and Genomics (ACMG).Ten DYSF gene variants (including 5 frameshift variants, 3 splicing variants, 1 missense variant and 1 nonsense variant) were detected. Among these, c.1375dupA (p.Met459Asnfs*15), c.610CT (p.Arg204X), c.1180+5GA and c.1284+2TC were known to be pathogenic, while c.4008_4010delCCTinsAC (p.Leu1337Argfs*8), c.1137_1169del (p.379_390del), c.754AG(p.Thr252Ala), c.1175_1176insGCAGAGTG (p.Met394Serfs*7), c.3114_3115insCGGC (p.Arg1040Profs*74) and c.1053+3GC were unreported previously. Of the six novel variants, c.1137_1169del, c.1175_1176insGCAGAGTG and c.3114_3115insCGGC were predicted as pathogenic (PVS1+PM2+PM3), c.4008_4010delCCTinsAC as likely pathogenic (PVS1+PM2), c.754AG and c.1053+3GC as variants of uncertain significance based on the ACMG standards and guidelines.Variants of the DYSF gene probably underlay Dysferlinopathy in the patients among the five pedigrees. Above finding has enriched the spectrum of DYSF gene variants.

To explore the genetic cause of a patient suspected for congenital ectodermal dysplasia with repeated hyperthermia and to assess the reproductive risk for his family.Medical whole-exome sequencing (WES) were used to detect single-nucleotide variations and low-coverage massively parallel copy number variation sequencing (CNV-seq) were employed to verify suspected CNVs. PCR and real-time quantitative PCR were applied to confirm the deletion of EDA gene.The results of WES suggested that the patient carried a hemizygous deletion for chrX:69 243 016-69 395 730. CNV-seq indicated that the patient carried a deletion of approximately 0.12 Mb on Xq13.1, which encompassed the EDA gene. The PCR results confirmed that there was a hemizygous deletion of exons 3 to 8 of the EDA gene. The same deletion was not found in his mother.The congenital ectodermal dysplasia of the patient may be attributed to deletion of exons 3 to 8 of the EDA gene, which could be de novo or derive from germline mosaicism of his mother. The WES and CNV-seq are of great value for the diagnosis of rare diseases.

To detect genomic copy number variations (CNVs) among 145 children with unexplained mental retardation/developmental delay (MR/DD) by using low-depth whole-genome copy number variation sequencing (CNV-seq).Peripheral blood samples were collected from the patients and subjected to DNA extraction and CNV-seq. The results were analyzed by a combination of bioinformatic tools.Forty-nine patients were found to carry a total of 67 CNVs with an average size of 5.27 Mb. Among these, 22 patients were assessed to carry MR/DD-related CNVs involving 21 syndromes. This gave a diagnostic rate of 15.17%(22/145) for CNVs associated with unexplained MR/DD. The corresponding regions of the 22 MR/DD-related CNVs in the human genome covered 174 MR/DD-related pathogenic genes, which have mapped to 18 sections on 10 chromosomes.Genomic CNVs-related microdeletions/duplications account for a significant proportion of unexplained MR/DD, for which CNV-seq can provide an accurate diagnosis.

Background Haemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the coagulation factor Ⅷ (F8) gene. Its incidence in men is estimated to be approximately 1/5000. Objective This study aimed to characterize the mutation spectrum of the F8 gene in 485 Chinese families, encompassing all HA phenotypic classes. Additionally, we evaluated the accuracy of prenatal diagnosis of foetuses at risk of having HA. Methods Long-Distance PCR (LD-PCR) and Multiplex PCR were used to detect inversions, next-generation sequencing (NGS) was used for point mutations, and multiplex ligation-dependent probe amplification (MLPA) was used for large deletions or duplications. Results A mutation spectrum of 478 HA families was produced. Throughout 26 exons and 15 introns, a total of 237 different alterations of mutations were detected, of which 146 are known mutations (64.5%) and 91 are novel mutations (35.5%). Prenatal diagnosis revealed 97 normal males (35.79%), 103 HA males (38.01%), 36 normal females (13.28%), and 38 HA carrier females (14.02%). Conclusion Using a systematic approach comprised of three steps, 237 pathogenic variants in 478 out of 485 patient samples (98.6%) were detected, including the identification of a heterogeneous mutation spectrum of 91 novel mutations. In addition, prenatal diagnosis of HA in pregnant carriers allowed for accurate determination of the foetal F8 gene state.

To explore the genetic etiology of three pedigrees with a gestational history of fetal renal anomalies.Peripheral venous blood or skin samples were derived from the probands of the three pedigrees. Copy number variation sequencing (CNV-seq) was applied to detect alterations of genome CNVs.The patient from pedigree 1 and the fetuses from pedigrees 2 and 3 all carried a heterozygous 17q12 deletion, with the size ranging from 1.4 Mb to 1.48 Mb encompassing the HNF1B gene.The diagnosis of 17q12 microdeletion may be difficult during fetal period for its variable phenotypes. Alterations of chromosomal copy numbers need to be excluded in such patients.

To assess the value of chromosomal microarray analysis (CMA) for the analysis of 824 samples from miscarriage or stillbirth.Copy number variations (CNVs) in the abortic chorionic villi or stillbirth tissues were detected by CMA.All specimens were successfully analyzed, among which 381 (46.2%) were diagnosed with chromosomal abnormalities, which included 312 (81.9%) numerical abnormalities, 66 (17.3%) structural abnormalities and 3 (0.8%) uniparental disomies. Among numerical chromosomal abnormalities, aneuploidies was most common (92.0%), with trisomy 16 and 45,X accounting for 41 (13.1%) and 63 (20.2%) of the cases, respectively. Among the 66 structural chromosomal aberrations, there were 26 (39.4%) CNVs duplications, 20 (30.3%) CNVs deletions, and 20 (30.3%) CNVs duplication and deletions. 33 CNVs were predicted as have a high chance to lead to a disease.CMA is a reliable, robust, and high-resolution method for the analysis of miscarriage or stillbirth samples. Numerical aberrations, in particular chromosomal aneuploides, are the main cause for spontaneous abortions and stillbirths.

Hermansky-Pudlak syndrome is a rare, autosomal, recessive syndromic form of albinism characterized by oculocutaneous albinism, bleeding diathesis, and a series of clinical complications. It is rarely reported in China, even with its large population base. In this study, we describe the clinical phenotypes and genotypes of five unrelated Chinese Hermansky-Pudlak syndrome pedigrees following clinical observation and next-generation sequencing. We identified three HPS-1 and two HPS-6 cases among 548 Chinese patients with oculocutaneous albinism. Five novel variants [c.1279_1280insGGAG p.(Asp427Glyfs*27) and c.875_878delACAG p.(Asp292Alafs*38) in HPS1 and c.1999C>T p.(Arg667*), c.335G>A p.(W112*), and c.1732C>T p.(R578*) in HPS6] were identified by next-generation sequencing. Our findings expand the spectrum of known variants and the genetic background of Hermansky-Pudlak syndrome, which may help in investigating phenotype-genotype relationships and aid in genetic counselling of patients with Hermansky-Pudlak syndrome.

Objective To evaluate the application value and significance of low-depth whole-genome sequencing for copy number variations (CNV-Seq) in the genetic diagnosis and prenatal diagnosis of X-linked ichthyosis (XLI) due to STS gene deletion. Methods Clinical data were collected from 3 616 subjects who received CNV-Seq, and single-gene test results were collected from 7 patients or pedigrees with ichthyosis in The First Affiliated Hospital of Zhengzhou University in 2018. The 3 616 samples included 2 891 prenatal samples from pregnant women (most were amniotic fluid samples, some fetal villus samples, very few umbilical blood samples) and 725 peripheral blood samples from other subjects. Genomic DNA was extracted from amniocytes or peripheral blood, and then subjected to CNV-Seq. Quantitative PCR (qPCR) and single nucleotide polymorphism (SNP) -comparative genomic hybridization (CGH) array were performed to verify the detected CNVs. Pathogenicity of the CNVs was analyzed according to the database of genomic variants (DGV) , database of genomic variation and phenotype in humans using ensembl resources (DECIPHER) , clinical genome resource (ClinGen) and online Mendelian inheritance in man (OMIM) . Results Of the 3 616 subjects receiving CNV-Seq, Xp22.31 deletion was identified in prenatal samples from 6 pregnant women, including 5 male and 1 female fetuses. The deleted fragment of Xp22.31 covered the XLI region containing the major gene STS. The parental CNV-Seq showed that the Xp22.31 deletion was spontaneous mutation in 2 of the 6 fetuses, and inherited from the parents in the other 4 fetuses. qPCR confirmed that the female fetus was a carrier of a complete heterozygous deletion of the STS gene, and there was a complete deletion of the STS gene in the other 5 male fetuses. SNP-CGH array also confirmed that the female fetus was heterozygous Xp22.31 deletion carrier, which was consistent with the CNV-Seq results. Ichthyosis gene panel sequencing in the 7 patients with ichthyosis showed 1 with harlequin ichthyosis, 2 with ichthyosis vulgaris, 3 with XLI, and no causative mutation in 1. CNV-Seq confirmed that Xp22.31 deletion existed in the above 2 patients with XLI due to STS gene deletion. Moreover, Xp22.31 duplication was found in 16 out of 3 616 subjects receiving CNV-Seq, but they were all individuals or fetuses with normal phenotype. Conclusions CNV-Seq is a stable and reliable method for screening whole-genome CNVs, and can be applied to genetic diagnosis and prenatal diagnosis of XLI due to STS gene deletion. The deletion of Xp22.31 fragment containing the STS gene can cause XLI, and the duplication of the same region is highly likely to be the polymorphic variation. Key words: Ichthyosis, X-linked; DNA copy number variations; Gene deletion; Sequence analysis, DNA; Prenatal diagnosis; Xp22.31 deletion

MAPKs play important roles in platelet activation. However, the molecular mechanisms by which MAPKs are regulated in platelets remain largely unknown. Real-time polymerase chain reaction and western blot data showed that MEKK3, a key MAP3K family member, was expressed in human and mouse platelets. Then, megakaryocyte/platelet-specific MEKK3-deletion (MEKK3-/- ) mice were developed to elucidate the platelet-related function(s) of MEKK3. We found that agonist-induced aggregation and degranulation were reduced in MEKK3-/- platelets in vitro. MEKK3 deficiency significantly impaired integrin αIIbβ3-mediated inside-out signaling but did not affect the outside-in signaling. At the molecular level, MEKK3 deficiency led to severely impaired activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun NH2-terminal kinase 2 but not p38 or ERK5. In vivo, MEKK3-/- mice showed delayed thrombus formation following FeCl3-induced carotid artery injury. Interestingly, the tail bleeding time was normal in MEKK3-/- mice. Moreover, MEKK3-/- mice had fewer microthrombi, reduced myocardial infarction (MI) size, and improved post-MI heart function in a mouse model of MI. These results suggest that MEKK3 plays important roles in platelet MAPK activation and may be used as a new effective target for antithrombosis and prevention of MI expansion.

Objective— MAPKs (mitogen-activated protein kinases), especially p38, play detrimental roles in cardiac diseases and cardiac remodeling post-myocardial infarction. However, the activation and function of MAPKs in coronary thrombosis in vivo and its relationship with clinical outcomes remain poorly understood. Approach and Results— Here, we showed that p38α was the major isoform expressed in human and mouse platelets. Platelet-specific p38α-deficient mice presented impaired thrombosis and hemostasis but had improved cardiac function, reduced infarct size, decreased inflammatory response, and microthrombus in a left anterior descending artery ligation model. Signaling analysis revealed that p38 activation was one of the earliest events in platelets after treatment with receptor agonists or reactive oxygen species. p38α/MAPK-activated protein kinase 2/heat shock protein 27 and p38α/cytosolic phospholipases A2 were the major pathways regulating receptor-mediated or hydrogen peroxide-induced platelet activation in an ischemic environment. Moreover, the distinct roles of ERK1/2 (extracellular signal–regulated kinase) in receptor- or reactive oxygen species-induced p38-mediated platelet activation reflected the complicated synergistic relationships among MAPKs. Analysis of clinical samples revealed that MAPKs were highly phosphorylated in platelets from preoperative patients with ST-segment–elevation myocardial infarction, and increased phosphorylation of p38 was associated with no-reflow outcomes. Conclusions— We conclude that p38α serves as a critical regulator of platelet activation and potential indicator of highly thrombotic lesions and no-reflow, and inhibition of platelet p38α may improve clinical outcomes in subjects with ST-segment–elevation myocardial infarction.

Dynamic endocytic and exocytic trafficking of integrins is an important mechanism for cell migration, invasion, and cytokinesis. Endocytosis of integrin can be classified as clathrin dependent and clathrin independent manners. And rapid delivery of endocytic integrins back to the plasma membrane is key intracellular signals and is indispensable for cell movement. Integrin αIIbβ3 plays a critical role in thrombosis and hemostasis. Although previous studies have demonstrated that internalization of fibrinogen-bound αIIbβ3 may regulate platelet activation, the roles of endocytic and exocytic trafficking of integrin αIIbβ3 in platelet activation are unclear. In this study, we found that a selective inhibitor of clathrin-mediated endocytosis pitstop 2 inhibited human platelet spreading on immobilized fibrinogen (Fg). Mechanism studies revealed that pitstop 2 did not block the endocytosis of αIIbβ3 and Fg uptake, but inhibit the recycling of αIIbβ3 to plasma membrane during platelet or CHO cells bearing αIIbβ3 spreading on immobilized Fg. And pitstop 2 enhanced the association of αIIbβ3 with clathrin, and AP2 indicated that pitstop 2 inhibit platelet activation is probably due to disturbance of the dynamic dissociation of αIIbβ3 from clathrin and AP2. Further study demonstrated that Src/PLC/PKC was the key pathway to trigger the endocytosis of αIIbβ3 during platelet activation. Pitstop 2 also inhibited platelet aggregation and secretion. Our findings suggest integrin αIIbβ3 trafficking is clathrin dependent and plays a critical role in platelet spreading, and pitstop 2 may serve as an effective tool to address clathrin-mediated trafficking in platelets.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.

Introduction: Many of the immunoglobulin (Ig) super family proteins are expressed on platelet surface, and involve in the regulation of platelet activation. Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptors LILRB2 or LILRB3, bearing extracellular Ig-like domains and immunoreceptor tyrosine-based inhibitory motifs, is broadly expressed and plays important roles in immune responses, neuron axonal regeneration and hematopoietic processes. The expressions and functions of PIRB in platelet remain unknown. Hypothesis: PIRB regulates platelet activation. Methods: PIRB intracellular domain deletion mice (PIRB-TM) were used to elucidate the functions of PIRB in platelet activation. Results: We found that LILRB2 and LILRB3 (or PIRB) expressed in human (or mouse) platelets. The Increasing megakaryocytes ratio in bone marrow and mild thrombocythemia were observed in PIRB-TM mice. Deficiency of PIRB facilitated agonists-induced platelet aggregation and spreading on immobilized fibrinogen (Fg). The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also enhanced. Further signaling analysis revealed that PIRB bound to SHP1 and SHP2. And collagen related peptide (CRP)-induced tyrosine phosphorylation of LAT, SLP76 and PLCγ2 increased in PIRB-TM platelet. The phosphorylation of FAK Y397 and integrin β3 Y785, but not integrin β3 Y773, was also enhanced in PIRB-TM platelet spread on immobilized Fg. We also found that one of the PIRB ligands angiopoietin like protein 2 (Angptl2) was highly expressed and stored in platelet α-granule, and was able to be released and attached to platelet surface upon platelet activation. Purified Angptl2 inhibited agonists-induced human platelet aggregation and spreading on immobilized Fg. Angptl2 inhibited CRP-induced phosphorylation of LAT, SLP76 and PLCγ2, and spreading-driven phosphorylation of FAK Y397 and integrin β3 Y785 in platelet. Conclusions: These results revealed that PIRB and its ligand Angptl2 attenuated platelet activation by suppression of GPVI-mediated and integrin αIIbβ3 mediated outside-in signaling. Therefore, LILRB2 and LILRB3 may prove to be the new promising antithrombotic targets.