Comprehensive analysis of genomic complexity in the 5' end coding region of the DMD gene in patients of exons 1-2 duplications based on long-read sequencing.

BACKGROUND: Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic variants in the DMD gene, however, 5' end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS), are commonly used in clinics. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation. RESULTS: We used LRS technology to perform whole genome sequencing on three families and analyze the structural variations of the DMD gene, which involves the duplications of exon 1 and/or exon 2. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. In pedigree 1, the male individuals harboring duplication variant of consecutive exons 1-2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is likely benign. In pedigree 3, the patient carries complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified by LRS, enabling the classification of the variants' pathogenicity. CONCLUSIONS: Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.

Mutations in PLCZ1 induce male infertility associated with polyspermy and fertilization failure.

PURPOSE: To investigate the genetic causes of polyspermy and total fertilization failure (TFF) in two independent male patients suffering from male infertility. METHODS: Immunofluorescence (IF) staining was used to detect the localization of the PLCζ protein in sperm and the maternal pronucleus in the zygote. Genomic DNA samples were extracted from the peripheral blood of patients and their families. The ExAC database was used to identify the frequency of corresponding mutations. The PLCZ1 mutations were validated by Sanger sequencing. The pathogenicity of the identified mutations and their possible effects on the protein were assessed using in silico tools and molecular modeling. RESULTS: We identified a reported homozygous mutation c.588C > A (p.Cys196Ter) and a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in PLCZ1. The IF results showed that these multipronuclear zygotes formed as a result of polyspermy. In silico analysis predicted that the mutations result in disease-causing proteins. IF staining revealed that PLCζ is abnormally localized in the sperm samples from the two affected patients. Assisted oocyte activation (AOA) successfully rescued polyspermy and TFF and achieved pregnancy in two patients with the PLCZ1 mutation. CONCLUSION: We identified a homozygous mutation in PLCZ1 (c.588C > A [p.Cys196Ter]) in a male patient with polyspermy after in vitro fertilization (IVF) as well as a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in a male patient with fertilization failure after intracytoplasmic sperm injection (ICSI), and we provide evidence that the homozygous mutation can cause polyspermy and the compound heterozygous mutation can cause fertilization failure.

A deep learning-based recognition for dangerous objects imaged in X-ray security inspection device.

Several limitations in algorithms and datasets in the field of X-ray security inspection result in the low accuracy of X-ray image inspection. In the literature, there have been rare studies proposed and datasets prepared for the topic of dangerous objects segmentation. In this work, we contribute a purely manual segmentation for labeling the existing X-ray security inspection dataset namely, SIXRay, with the pixel-level semantic information of dangerous objects. We also propose a composition method for X-ray security inspection images to effectively augment the positive samples. This composition method can quickly obtain the positive sample images using affine transformation and HSV features of X-ray images. Furthermore, to improve the recognition accuracy, especially for adjacent and overlapping dangerous objects, we propose to combine the target detection algorithm (i.e., the softer-non maximum suppression, Softer-NMS) with Mask RCNN, which is named as the Softer-Mask RCNN. Compared with the original model (i.e., Mask RCNN), the Softer-Mask RCNN improves by 3.4% in accuracy (mAP), and 6.2% with adding synthetic data. The study result indicates that our proposed method in this work can effectively improve the recognition performance of dangerous objects depicting in the X-ray security inspection images.

[Effect of complete Y chromosome AZFc microdeletion on embryo euploidy].

OBJECTIVE: Preimplantation genetic testing (PGT) was performed to analyze the embryo euploidy in patients with complete Y chromosome AZFc microdeletion. METHODS: The clinical data of complete AZFc microdeletion underwent PGT from January 2013 to December 2021 in Reproductive Medicine Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively analyzed. The patients with monogenic disease who underwent PGT during the same period were set as the control group. The basic characteristics, fertilization rate, Day 3 high quality embryo rate, blastocyst formation rate, embryo euploid rate, 45, X embryo ratio was compared between the two groups. RESULTS: A total of 220 patients were included, including 91 patients with complete AZFc microdeletion and 129 patients with monogenic disease. There was no significant difference in age between the two groups. In semen parameters, the sperm concentration, total sperm count and progressive motility in AZFc microdeletion group were lower than those in monogenic disease group, and the differences were statistically significant (P=0.001). The fertilization rate of AZFc microdeletion group was lower than that in monogenic disease group (P=0.012), and there was no significant difference in the number of MII oocytes, Day 3 high-quality embryo rate and blastocyst formation rate. A total of 933 blastocysts were successfully tested, including 496 blastocysts in AZFc deletion group and 437 blastocysts in monogenic disease group. The euploid, aneuploid and mosaic rates of the AZFc microdeletion group were 57.26%, 24.60% and 18.14%, respectively, while those of the monogenic disease group were 66.13%, 23.80% and 10.07%, with statistically significant differences between the two groups (P=0.001). Further analysis of the two groups of aneuploid embryos showed that aberrations occurred most commonly in chromosome16 (3.87%), X (3.46%), 13 (2.44%), 22 (2.24%) and 19 (2.03%) in AZFc microdeletion group, respectively, while the monogenic disease group was 22 (4.35%), 16 (2.97%), 7 (2.74%), 15(1.60%) and 2(1.60%), respectively. The proportion of sex chromosome abnormality in AZFc microdeletion group was higher than that in monogenic disease group (P=0.039), and there was no significant difference in the proportion of 45,X between the two groups. CONCLUSION: Compared with monogenic disease group, The embryo euploid rate in AZFc microdeletion patients decreased and the proportion of 45, X embryos did not increase significantly. It is recommended to select euploid female embryos by PGT, which not only avoids vertical transmission of AZFc microdeletion, but also reduces the risk of miscarriage due to aneuploid embryos.

Nucleoporin37 may play a role in early embryo development in human and mice.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.

Majority of transferred mosaic embryos developed healthy live births revealed by a preclinical study using embryonic morphology assessment and noninvasive PGT-A on cell-free DNA in blastocoel fluid.

PURPOSE: This preclinical study aimed to evaluate whether using transferred mosaic embryos (primarily selected by embryonic morphology assessment (EMA) and compared by the noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) on cell-free DNA in blastocoel fluid (BF)) increases the rates of clinical pregnancies (CPs) and healthy live births (HLBs) and to investigate whether niPGT-A could provide valuable genetic information for the EMA-selected transferred mosaic embryos. METHODS: This study collected 215 blastocyst culture samples and 182 BF samples. Cell-free DNA from the BF was amplified and examined by next-generation sequencing-based niPGT-A. All 182 patients underwent EMA. However, only 147 underwent in vitro fertilization and embryo transfer, and only 113 clinical outcomes were followed up. Comprehensive chromosome screening for the chorionic villus sampling of spontaneous miscarriages and noninvasive prenatal testing for ongoing pregnancies were also performed. RESULTS: The implantation rate was 77.55% in 147 transferred high-quality embryos selected by EMA. Among 113 CPs, 16 led to spontaneous miscarriage (14.16%), and 97 resulted in HLBs (85.84%). According to the niPGT-A results for 113 patients with clinical outcomes, 80.4% had CP (euploid, 20.54%; single aneuploid, 1.79%; mosaic chromosome aneuploid and/or segmental aneuploid, 58.04%). Of all the mosaic aneuploids, 90.76% were false positive, transforming to euploid. CONCLUSIONS: Transferred EMA-selected embryos showed higher implantation rates. The niPGT-A of BF provided valuable genetic status ("-ploid") information, which helped reduce aneuploid-induced implantation failure and miscarriage, thereby increasing the CP and HLB rates. Additionally, majority of the transferred embryos with complex/chaotic mosaic aneuploid would likely develop HLBs.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

BACKGROUND: Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS: Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION: Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

The role of cathepsin L on structural changes of collagen fibers involved in textural deterioration of chilled grass carp (Ctenopharyngodon idella) fillets.

BACKGROUND: Textural deterioration is a serious problem in chilled fish flesh. Cysteine proteinases are proposed to participate in disintegration of collagen fibers during this process, while its mechanism remains elusive. In the present study, a cysteine proteinase was purified from grass carp muscle and identified by mass spectrometry, and its effect on structural changes of collagen fibers was investigated. RESULTS: During storage at 4 °C, cysteine proteinase activity in fillets increased to 1.53-fold at day 5 and maintained a high level later, and this variable was related to a decline in shear force and an increase in drip loss. A 29 kDa cysteine proteinase was purified through ammonium sulfate precipitation and column chromatography, and identified as cathepsin L. Cathepsin L caused collagen fibers to partly disintegrate into fibril bundles and individual fibrils at 48 h, while the triple helical structure of collagen molecules remained stable. Release of soluble proteins and glycosaminoglycans from cathepsin L-treated collagen fibers was time dependent, coinciding with a release of 4.12 ± 0.13% and 8.57 ± 0.03% at 48 h respectively. However, 0.85 ± 0.02% of hydroxyproline was freed from cathepsin L-treated collagen fibers at 48 h. Furthermore, scanning electron microscopy revealed that the inhibitory effect of cathepsin L could retard the destruction of intramuscular connective tissues (IMCTs). CONCLUSION: These results indicated that cathepsin L might be involved in collagen fiber breakdown by degrading collagen-associated proteoglycans during textural deterioration of grass carp. © 2022 Society of Chemical Industry.

[Analysis of three Chinese pedigrees affected with recurrent hydatidiform mole due to variants of NLRP7 gene].

OBJECTIVE: To explore the genetic etiology of recurrent hydatidiform mole (RHM) and provide accurate guidance for reproduction. METHODS: Peripheral venous blood samples of the probands with RHM and members from 5 unrelated pedigrees were collected. Genomic DNA was extracted by using routine method, and whole exome sequencing was carried out to detect variants of RHM-associated genes including NLRP7 and KHDC3L. Sanger sequencing and real-time quantitative PCR (RT-qPCR) were used to validate the candidate variants and delineate their parental origin. RESULTS: Homozygous or compound heterozygous variants of the NLRP7 gene were identified in four patients from three pedigrees, which included a homozygous deletion of exon 1 to 4 of NLRP7 in patient P1 and her elder sister, compound heterozygous variants of NLRP7 c.939delG (p.Q314Sfs*6) pat and c.1533delG (p.N512Tfs*4) mat in patient P2, and compound heterozygous variants of NLRP7 c.2389_2390delTC (p.A798Qfs*6) pat and c.2165A>G (p.D722G) mat in patient P4. All variants were interpreted as pathogenic or likely pathogenic according to the American College of Medical and Genomics (ACMG) guidelines. Among these, NLRP7 exons 1 to 4 deletion, c.939delG (p.Q314Sfs*6), c.1533delG (p.N512Tfs*4) and c.2389_2390delTC (p.A798Qfs*6) were unreported previously. CONCLUSION: Variants of the NLRP7 gene probably underlay autosomal recessive RHM in the three pedigrees, and definitive molecular diagnosis is beneficial for accurate genetic counseling. Above finding has also enriched the spectrum of the NLRP7 variants underlying RHM.

Multicolor Zinc(II)-Coordinated Hydrazone-Based Bistable Photoswitches for Rewritable Transparent Luminescent Labels.

Repeatable printing of invisible multicolor luminescence patterns with long retention times on transparent substrates is of significant importance, but it remains a formidable challenge. Here, two novel hydrazone-based on/off fluorescent photoswitches with decent emission quantum efficiencies, good reversible photoisomerization properties, and extremely long thermal half-lives, were designed and synthesized. Excitingly, X-ray crystallography data of both Z and E isomers of one hydrazone-based photoswitch were obtained. Multiple emission colors of blue, cyan, green, yellow, and orange can be obtained readily for these two photoswitches upon coordination with various zinc salts. Moreover, the photo-controlled rewritable printing of invisible multicolor images with high resolution was achieved by using these photoswitches. Importantly, the legibility of the printed patterns can last at least over 3 months without detectable emission intensity loss under ambient conditions.

Phosphorescent Soft Salt Based on Platinum(II) Complexes: Photophysics, Self-Assembly, Thermochromism, and Anti-counterfeiting Application.

A new platinum(II) complex-based soft salt S1, ([Pt(tpp)(ed)]+[Pt(pba) (CN)2]-) (tpp = 2-(4-(trifluoromethyl)phenyl)pyridine, ed = ethane-1,2-diamine, pba = 4-(2-pyridyl)benzaldehyde), was designed and synthesized. UV-visible absorption and photoluminescence (PL) spectra were studied to elucidate the nature of ground and excited states. The soft salt complex was found to show self-assembly properties with the assistance of electrostatic, π-π stacking, and Pt···Pt interactions, resulting in the remarkable emergence of low-energy absorption and PL bands. Morphological transformation of S1 from undefined nanosized aggregates to nanofibers with different solvent compositions has been demonstrated. Interestingly, a luminescent polymer film was prepared by doping S1 into a polyethylene glycol matrix. The film displayed distinctive emission color change from yellow to red upon heating. Eventually, a high-level anti-counterfeiting application was accomplished using a time-resolved imaging technique based on the thermochromic luminescence property and long emission decay time displayed by S1. It is anticipated that this work can provide deep insights into the control of intermolecular interactions between cationic and anionic complexes of soft salt upon exposure to different external stimuli, resulting in the development of smart luminescent materials for various applications.

Genetic etiologic analysis in 74 Chinese Han women with idiopathic premature ovarian insufficiency by combined molecular genetic testing.

PURPOSE: To identify the disease-causing genes of Chinese Han women with idiopathic premature ovarian insufficiency (POI). METHODS: Seventy-four Chinese Han women with idiopathic POI were collected to analyze the genetic etiology. Triplet repeat-primed polymerase chain reaction (TP-PCR) was performed to screen the FMR1 (CGG)n premutation, and then 60 POI-related genes were sequenced by targeted next-generation sequencing (NGS) in POI patients with normal FMR1. RESULTS: A total of one patient (1/74) with FMR1 premutation was identified. Targeted NGS revealed that 15.07% (11/73) patients had pathogenic or likely pathogenic variants of Mendelian genes (FOXL2, EIF2B2, CYP17A1, CLPP, MCM9, GDF9, MSH5, ERCC6, POLG). Ten novel variants in six Mendelian genes were identified, such as CLPP c.355A>C (p.I119L) and c.688A>C (p.M230L), MCM9 c.1157C>T (p.T386M) and c.1291A>G (p.M431V), GDF9 c. 238C>T (p.Q80X), MSH5 c.604G>C (p.G202R) and c.2063T>C (p.I688T), ERCC6 c.C1769C>T (p.P590L), POLG c.2832G>C (p.E944D), and c.2821A>G (p.I941V). CONCLUSION: This study suggested targeted NGS was an efficient etiologic test for idiopathic POI patients without FMR1 premutation and enriched the variant spectrum of POI-related genes.

The fertility willingness and acceptability of preimplantation genetic testing in Chinese patients with autosomal dominant polycystic kidney disease.

BACKGROUND: With the development and progression of genetic technology, preimplantation genetic testing (PGT) has made it possible to block the inheritance of autosomal dominant polycystic kidney disease (ADPKD) as early as possible. However, we need to know the patients' fertility intentions and their acceptance of PGT. METHODS: A questionnaire survey was conducted to collect data on the basic demographic data, quality of life, social support, fertility willingness, and level of understanding of genetic testing for blocking the inheritance of ADPKD among patients aged 18-45 years in seven hospitals from January 2018 to December 2018. After verification, statistics were calculated. RESULTS: A total of 260 patients with ADPKD were interviewed, including 137males (52.7%) and 123 females (47.3%). The overall fertility willingness rate was low (n = 117, 45.0%). The proportion of married patients aged 25-34 years that were at the optimal reproductive age but did not yet have children was relatively high (n = 77, 67.0%). The fertility intentions of ADPKD patients were significantly influenced by age (OR: 0.101, 95% CI 0.045-0.225, P < 0.001) and education level (OR: 2.134, 95% CI 1.162-3.917, P = 0.014). Among patients who are willing to have children, 207 (79.6%) of them would choose PGT technology. Among those who were not sure whether they would choose PGT technology, the first major concern was technical safety (49.2%). CONCLUSIONS: The reproductive desire of childbearing ADPKD patients in China was low. Strengthening the health education of ADPKD genetic knowledge and reducing the cost of related technologies may improve the fertility intentions and reduce the barriers to acceptance of PGT.

Preimplantation genetic testing for complex chromosomal rearrangement carriers by next-generation sequencing.

RESEARCH QUESTION: Can preimplantation genetic testing (PGT) with next-generation sequencing (NGS) increase the chance of achieving a balanced euploid pregnancy in complex chromosome rearrangement (CCR) carriers? DESIGN: Six couples underwent PGT at the Clinical Centre of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University. The CCR carriers in the six couples were: Case A: 46,XY,t(1;4;11)(p31;p16;q22); Case B: 46,XY,t(3;13;5)(p14;q21;p14); Case C: 46,XX,t(6;11;21)(q21;q21;q13); Case D: 46,XX,inv(9)(p12; q13),t(13;15)(q14;q24); Case E: 46,XX,inv(9)(p12;q13),t(7;9)(q22;p22); and Case F: 46,XX,t(2;7)(q21;q36),t(2;4)(p10;q10),t(2;4)(q15;q10). After ovarian stimulation followed by oocyte retrieval and embryo culture, PGT was performed on day 5 or 6 blastocyst biopsies using NGS to identify normal/balanced euploid embryos. Vitrified-warmed single embryo transfers were performed using normal/balanced euploid embryos. RESULTS: After seven cycles, 84 oocytes were retrieved. Whole genome sequencing by NGS was performed on 25 trophectoderm biosies. Six (24%) embryos were identified as normal/balanced euploid, four were transferred resulting in four live births. Case A, C, D and E each gave birth to a healthy baby after their first cycle. There was no transferable embryo after two cycles for Case B and one cycle for Case F. The implantation rate per transfer was 4/4 and the live birth rate was 4/4. CONCLUSION: These results strongly support the use of NGS for CCR carriers.

Homozygous nonsense mutation Trp28X in the LHB gene causes male hypogonadism.

PURPOSE: The purpose of this study was to investigate a novel mutation in the luteinizing hormone beta-subunit (LHB) gene in one male patient with hypogonadism due to selective luteinizing hormone (LH) deficiency. METHODS: Sanger sequencing of one 28-year-old man born to consanguineous parents was performed. Treatment with human chorionic gonadotropin (hCG) (2000 IU, twice a week) was initiated for 3 months, followed by 5000 IU weekly to date. RESULTS: We identified a novel c.84G>A[p.W28X] nonsense LHB mutation. The W28X mutation produces a truncated LHB peptide of seven amino acids, which prevents the synthesis of intact LH. After 40 days of treatment with hCG, the patient exhibited a few spermatozoa in the semen. Treated for 6 months, the patient exhibited normal seminal parameters. CONCLUSIONS: We identified a novel mutation in the LHB gene in a male patient with hypogonadism and provided evidence that LHB nonsense mutation can cause selective LH deficiency. We reconfirmed hCG treatment may restore male fertility due to LHB mutation.

[Preimplantation genetic diagnosis for infertile males with autosomal dominant polycystic kidney disease].

OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic renal diseases, which may cause oligoasthenospermia and azoospermia and result in male infertility. This study aimed to analyze the outcomes of preimplantation genetic diagnosis (PGD) in male patients with ADPKD-induced infertility. METHODS: We retrospectively analyzed the clinical data on 7 male patients with ADPKD-induced infertility undergoing PGD from April 2015 to February 2017, including 6 cases of oligoasthenospermia and 1 case of obstructive azoospermia, all with the PKD1 gene heterozygous mutations. Following intracytoplasmic sperm injection (ICSI), we performed blastomere biopsy after 5 or 6 days of embryo culture and subjected the blastomeres to Sureplex whole-genome amplification, followed by haplotype linkage analysis, Sanger sequencing, array-based comparative genomic hybridization to assess the chromosomal ploidy of the unaffected embryos, and identification of the unaffected euploid embryos for transfer. RESULTS: One PGD cycle was completed for each of the 7 patients. Totally, 26 blastocysts were developed, of which 12 were unaffected and diploid. Clinical pregnancies were achieved in 6 cases following 7 cycles of frozen embryo transplantation, which included 5 live births and 1 spontaneous abortion. CONCLUSIONS: For males with ADPKD-induced infertility, PGD may contribute to high rates of clinical pregnancy and live birth and prevent ADPKD in the offspring as well. This finding is also meaningful for the ADPKD patients with normal fertility.

Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone.

Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos.

Chromosome aneuploidies commonly arise in embryos produced by assisted reproductive technologies and represent a major cause of implantation failure and miscarriage. Currently, preimplantation genetic diagnosis (PGD) is performed by array-based methods to identify euploid embryos for transfer to the patient. We speculated that a combination of next-generation sequencing technologies and sophisticated bioinformatics would deliver a more comprehensive and accurate methodology to improve the overall efficacy of embryo testing. To meet this challenge, we developed a high-resolution copy number variation (CNV) sequencing pipeline suitable for single-cell analysis. In validation studies, we showed that CNV-Seq was highly sensitive and specific for detection of euploidy, aneuploidy, and segmental imbalances in 24 whole genome amplification samples from PGD embryos that were originally diagnosed by gold standard array comparative genomic hybridization. In addition, CNV-Seq was capable of detecting, mapping, and accurately quantifying terminal chromosome imbalances down to 1 Mb in size originating from abnormal segregation of translocation chromosomes. These validation studies indicate that CNV-Seq displays the hallmarks of an accurate and reliable embryo test with the potential to further improve the overall efficacy of PGD.

Successful PGD for late infantile neuronal ceroid lipofuscinosis achieved by combined chromosome and TPP1 gene analysis.

Late infantile neuronal ceroid lipofuscinosis (NCL-2) is a severe debilitating autosomal recessive disease caused by mutations in TPP1. There are no effective treatments, resulting in early childhood death. A couple with two affected children presented for reproductive genetic counselling and chose to undertake IVF and preimplantation genetic diagnosis (PGD) to avoid the possibility of another affected child. However, DNA testing revealed only one mutation in the proband inherited from mother. Linkage analysis identified five informative linked short tandem repeat markers to aid the genetic diagnosis. Following IVF, five cleavage-stage embryos were biopsied and blastomeres were first subjected to whole-genome amplification, then a series of down-stream molecular genetic analyses to diagnose TPP1 genotype and finally array comparative genomic hybridization (CGH) to assess the chromosomal ploidy of each embryo. Two unaffected euploid embryos were identified for transfer. One was transferred on day 5 resulting in an ongoing pregnancy. Confirmatory prenatal diagnosis by amniocentesis showed concordance of the embryo and fetal diagnosis. As far as is known, this is the first successful report of PGD for NCL-2 using double-factor PGD with simultaneous single-gene testing and array CGH to identify an unaffected and chromosomally normal embryo for transfer.

[Identification of a novel c.822 G>T mutation of EDA gene in a Chinese family with X-linked hypohidrotic ectodermal dysplasia].

OBJECTIVE: To identify potential mutation of ectodysplasin A (EDA) gene in a Chinese family affected with X-linked hypohidrotic ectodermal dysplasia. METHODS: Blood samples were collected from the affected male proband, his family members and 103 unrelated individuals. Following extraction of genomic DNA, coding sequence of the EDA gene was amplified with PCR, and DNA sequencing was performed to detect potential mutation. RESULTS: A novel missense mutation, c.822G>T (p.W274C), was identified in exon 7 of the EDA gene in the proband, whilst his mother was found to be a heterozygous carrier. The same mutation was also found in 5 other family members including one affected male and four females, but was absent in unaffected males and 103 unrelated individuals. CONCLUSION: A c.822G>T mutation in exon 7 of the EDA gene probably underlies the disease in this Chinese family.