Renal agenesis-related genes are associated with Herlyn-Werner-Wunderlich syndrome.

OBJECTIVE: To explore the genetic causes of Herlyn-Werner-Wunderlich syndrome (HWWS) using whole-exome sequencing. DESIGN: Retrospective genetic study. SETTING: Academic medical center. PATIENT(S): Twelve patients with HWWS. INTERVENTION(S): Whole-exome sequencing was performed for each patient. Sanger sequencing was used to confirm the potential causative genetic variants. In silico analysis and American College of Medical Genetics and Genomics guidelines were used to classify the pathogenicity of each variant. MAIN OUTCOME MEASURE(S): Rare sequence variants associated with müllerian duct development and renal agenesis were identified and included in subsequent analyses. RESULT(S): A total of 11 variants were identified in 10 of 12 patients (83.3%) and were considered to constitute a molecular genetic diagnosis of HWWS. These 11 variants were related to 9 genes: CHD1L, TRIM32, TGFBR3, WNT4, RET, FRAS1, FAT1, FOXF1, and PCSK5. All variants were heterozygous and confirmed by Sanger sequencing. The changes included one frameshift variant, one splice-site variant, and eight missense variants. All of the identified variants were absent or rare in Genome Aggregation Database East Asian populations. One of the 11 variants (9.1%) was classified as a pathogenic variant according to the American College of Medical Genetics and Genomics guidelines, and 8 of the 11 variants (72.7%) were classified as variants of uncertain significance. CONCLUSION(S): To our knowledge, this is the first report of the genetic causes of HWWS. Renal agenesis-related genes, such as CHD1L, TRIM32, RET, and WNT4, may be associated with HWWS. Identification of these variants can not only help us understand the etiology of HWWS and the relationship between reproductive tract development and urinary system development, but additionally improve the level of genetic counseling for HWWS.

Sequence variant in the CDC42BPB gene is potentially associated with Mullerian duct anomalies.

AIM: Mullerian duct anomalies (MDA) are common female genital tract malformations. Genetic and environmental factors are important causes of MDA in women. Although many genes and mutations have been found to be associated with the pathogenesis of MDA, in most cases, the genetic pathogenic factors of MDA are still unknown. METHODS: We first analyzed the three sisters using low coverage whole-genome sequencing. Then whole-exome sequencing was carried out in each patient. The identified sequence variant was confirmed by Sanger sequencing. In silico pathogenicity analysis and conservative analysis of the mutation site were also performed. Protein structural modeling was used to analyze the effect of the mutated amino acid. RESULTS: We first analyzed the three sisters with septate uterus using low coverage whole-genome sequencing, but no possible pathogenic copy number variation was found. Then whole-exome sequencing was performed on the three sisters, and a rare homozygous variant, CDC42BPB:c.2012G>A:p.R671Q, was identified. All three patients were found with this variant. Sanger sequencing validated that this variant was segregated within the family. In silico pathogenicity analysis and conservative analysis of the mutation site suggested that the variant might be damaging. Protein structural analysis suggested that R671Q might weaken the electrostatic potential of this region, which may be a significant regulation target or protein interaction surface of CDC42BPB. CONCLUSION: We demonstrated that CDC42BPB genetic variant might be potentially associated with the pathogenesis of MDA.

Whole-Exome Sequencing Identified a TBX6 Loss of Function Mutation in a Patient with Distal Vaginal Atresia.

STUDY OBJECTIVE: The purpose of this study was to determine if there are any genetic changes with whole-exome sequencing associated with distal vaginal atresia. DESIGN: This was a retrospective genetics study of 5 patients who presented with distal vaginal atresia who were recruited between 2017 and 2018. Whole-exome sequencing was performed in each subject with distal vaginal atresia. Sanger sequencing was used to confirm the potential causative genetic mutation. SETTING: Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China. PARTICIPANTS AND MAIN OUTCOME MEASURES: The main outcome measure was the rare mutations potentially associated with distal vaginal atresia in 5 patients. RESULTS: A truncating mutation c.266delC (p.P89Rfs*5) in the T-box transcription factor 6 (TBX6) gene, which is highly expressed in the human vagina, was identified in 1 patient using whole-exome sequencing. The deletion of the 16p11.2 region containing the TBX6 locus has also been reported previously to have the clinical feature of Müllerian agenesis. This mutation was paternally inherited by the patient. This truncating mutation was absent from all of the databases we checked, suggesting that the variant is rare and pathogenic. CONCLUSION: We showed, to our knowledge, for the first time, that the mutation in TBX6 might be associated with human distal vaginal atresia.

CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in patients on long-term treatment: HIV-1 p24-producing cells and suppression of anti-HIV immunity.

BACKGROUND: A major question when attempting to eradicate and treat HIV-1 infection is how to reactivate latent proviruses. Stimulating HIV-1-specific cytolytic T lymphocytes (CTL) has been shown to facilitate the elimination of the latent viral reservoir after viral reactivation. Regulatory T (Treg) cells are known to be capable of lowering both HIV-specific immunoreactions and general immune activation during HIV-1 infection. It was hypothesized that the depletion of Treg cells could increase the HIV-1-specific cytolytic T lymphocyte response and reactivate HIV-1 p24 production. METHODS: Treg cells were isolated by isolation kit according to the surface marker of Treg cells. Real-time PCR method was used to quantify HIV-1 DNA. P24 antigens in the cell culture supernatant was done by ELISA. Cells activation and HIV specific HIV-1 CD8+ T cells were analyses using a FACSCalibur flow cytometer and CELLQUEST software. RESULTS: This study included both HIV-infected patients who were antiviral treatment-naïve and patients with sustained viral responses to antiretroviral therapy (ART) for 1 or 5 years. It was found that the HIV-DNA levels in Treg cells were approximately 10-fold higher than those in non-Treg CD4+ cells and that the depletion of Treg cells could enhance the frequency of HIV-1-specific CTL and immune activation after 5 years of effective ART. CONCLUSIONS: CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in long-term ART patients. Treg cells may be a target for eliminating the latent HIV reservoir after effective long-term ART.

[Establishing a 29 Y-STR Loci Multiplex PCR System].

OBJECTIVE: To establish a 29 Y-STR loci multiplex PCR system for investigating the genetic polymorphisms and to assess its application value in forensic science. METHODS: A multiplex PCR system was established using a five color fluorescence labeling 29 Y-STR loci (DYS456, DYS389 I , DYS437, DYS447, DYS389 11, DYS438, DYS522, DYS460, DYS458, DYS622, DYS390, DYS392, DYS448, DYS449, DYS391, Y-GA TA-H4, DYS388, DYS19, DYS385a/b, DYS527a/b, DYS393, DYS459a/b, DYS635, DYS439, DYS570 and DYS627) for multiple amplification and capillary electrophoresis. And its applicability was validated with genetic polymorphism data of 29 Y-STR of unrelated 2,000 male samples in Shandong Han population. RESULTS: A total of 1,981 different haplotypes of 2,000 individuals showed genotype diver- sity between 0.370 0 and 0.965 4. The system provided stable and accurate typing with high sensitivity of 0.05 ng. It satisfied the needs of variety of routine biological samples. CONCLUSION: The 29 Y-STR loci multiplex PCR system could be applied for actual cases and establishment of Y-STR database. In addition, it has great significance in forensic science practices and related research.

[Analysis of the characteristics of side population cells in the human ovarian cancer cell line OVCAR-3].

OBJECTIVE: To identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells. METHODS: SP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342. Limiting dilution transplantation assay, real-time PCR, and drug sensitivity assay were performed to compare the tumorigenic ability, differentiation ability in vivo, the mRNA expression of "stemness" marker (Oct-4, Klf4, and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2, ABCB1, and ABCC2), and response to multiple drugs (cisplatin, paclitaxel, doxorubicin, and mitoxantrone) between SP and NSP cells. RESULTS: A few of SP cells [(1.13 ± 0.39)%] which were sensitive to reserpine were identified in OVCAR-3 cells. The injection of as few as 10(2) SP cells initiated tumors in two of five mice. Tumor latency was 52 - 61 days. However, the NSP cells did not generate any tumors in mice until 10(4) NSP cells were injected (two of five mice). Tumor latency was 64 - 98 days. Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells. The SP cells regenerated both SP [(2.09 ± 0.73)%] and NSP populations in vivo with a fraction size that was comparable to the original population. The mRNA expression of "stemness" genes Oct-4, Klf4 and ABC transporters ABCG2, ABCC2 genes were elevated in SP cells compared to NSP cells, the fold changes were 1.95 ± 0.41 (P < 0.05), 4.26 ± 0.63 (P < 0.01), 3.22 ± 0.36 (P < 0.01), and 1.76 ± 0.26 (P < 0.01), respectively. The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P < 0.01), 0.521 ± 0.092 versus 0.384 ± 0.073 (P < 0.05), 0.742 ± 0.051 versus 0.526 ± 0.088 (P < 0.01), and 0.690 ± 0.096 versus 0.466 ± 0.112 (P < 0.01) when they exposed to 0.25 µg/ml cisplatin, 0.01 µmol/L paclitaxel, 0.25 µmol/L doxorubicin, and 0.05 µg/ml mitoxantrone, respectively. CONCLUSIONS: SP cells from OVCAR-3 have enhanced self-renewal, differentiation, and tumor-initiating capacity compared to NSP cells. The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells, which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells. Therefore, SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.

The effect of pedicle screw insertion through the neurocentral cartilage on the growth of immature canine vertebra.

OBJECTIVE: To explore the influence of pedicle screw insertion through the neurocentral cartilage (NCC) on the development of vertebrae and spinal canal in an animal experiment. METHODS: Sixteen dogs were randomly assigned to three groups: in group 1, posterior muscles at the surgery site were dissected; in group 2, the pedicles were drilled through the NCC by screws; in group 3, screws were placed in the pedicles through the NCC. Vertebrae of T8, T10, T12, L2, L4 and L6 were studied with the average data of the adjacent two vertebrae serving as controls. Spiral computerized tomography (CT) was used to assess the morphologic parameters of studied vertebrae and their controls. Measurements were made by an independent radiologist on the first post-operative day and 3 months after operation. Paired Student's t-tests of studied vertebrae and their controls were performed to evaluate the effect of pedicle screw insertion. RESULTS: In group 3, 3 months after operation the area, transverse diameter and anterior-posterior diameter of the vertebral canal and length of pedicle of studied vertebrae were significantly smaller than those of control vertebrae (P < 0.05). There were no significant differences in morphologic parameters between the studied vertebrae and the control vertebrae in groups 1 and 2 (P > 0.05). CONCLUSION: (i) Pedicle screw placement has a significant impact on the growth of the canine vertebra canal, and may lead to iatrogenic spinal stenosis, but their placement has no significant effect on the vertebral bodies; and (ii) the NCC can repair itself automatically. Drilling pedicle bone through the NCC with a screw and then removing the screw has no obvious impact on the growth of vertebrae.