Target Region Capture Sequencing Research Articles

Novel PSEN1 (P284S) Mutation Causes Alzheimer's Disease with Cerebellar Amyloid β-Protein Deposition.

AD-associated PSEN1 mutations exhibit high clinical heterogeneity. The discovery of these mutations and the analysis of their associations with cases such as EOAD should be critical to understanding AD's pathogenesis. We performed clinical analysis, neuroimaging, target region capture and high-throughput sequencing, and Sanger sequencing in a family of 3 generations. The underlying Alzheimer's pathology was evaluated using biomarker evidence obtained from cerebrospinal fluid (CSF) amyloid testing and 18F-florbetapir (AV-45) PET imaging. Target region capture sequencing revealed a novel heterozygous C to T missense point mutation at the base position 284 (c.850 C>T) located in exon 8 of the PSEN1 gene, resulting in a Prolineto- Serine substitution (P284S) at codon position 850. The mutation was also identified by Sanger sequencing in 2 family members, including proband and her daughter and was absent in the other 4 unaffected family members and 50 control subjects. Cerebrospinal fluid (CSF) amyloid test exhibited biomarker evidence of underlying Alzheimer's pathology. 18F-florbetapir (AV-45) PET imaging indicated extensive cerebral cortex and cerebellar Aβ deposition. We discovered a novel PSEN1 pathogenic mutation, P284S, observed for the first time in a Chinese family with early-onset AD.

Using Both Plasma and Urine Donor-Derived Cell-Free DNA to Identify Various Renal Allograft Injuries.

This study was designed to investigate the association between donor-derived cell-free DNA (dd-cfDNA) and renal allograft injuries. This single-center study enrolled 113 adult kidney transplant recipients with kidney biopsies. Plasma and urine dd-cfDNA was detected by target region capture sequencing. Plasma dd-cfDNA fraction was increased in multiple types of injuries, but most significantly in antibody-mediated rejection. Plasma dd-cfDNA fraction in isolated antibody-mediated rejection (1.94%, IQR: 1.15%, 2.33%) was higher than in T cell-mediated rejection (0.55%, IQR: 0.50%, 0.73%, P = 0.002) and negative biopsies (0.58%, IQR: 0.42%, 0.78%, P < 0.001), but lower than in mixed rejection (2.49%, IQR: 1.16%, 4.90%, P = 0.342). Increased urine dd-cfDNA concentration was associated with several types of injury, but most significantly with BK polyomavirus-associated nephropathy. Urine dd-cfDNA concentration in BK polyomavirus-associated nephropathy (12.22 ng/mL, IQR: 6.53 ng/mL, 31.66 ng/mL) was respectively higher than that in T cell-mediated rejection (5.24 ng/mL, IQR: 3.22 ng/mL, 6.99 ng/mL, P = 0.001), borderline change (3.93 ng/mL, IQR: 2.45 ng/mL, 6.30 ng/mL, P < 0.001), and negative biopsies (3.09 ng/mL, IQR: 1.94 ng/mL, 5.05 ng/mL, P < 0.001). Plasma dd-cfDNA fraction was positively associated with glomerulitis (r = 0.365, P < 0.001) and peri-tubular capillaritis (r = 0.344, P < 0.001), while urine dd-cfDNA concentration correlated with tubulitis (r = 0.302, P = 0.002). Both plasma and urine dd-cfDNA are sensitive markers for renal allograft injuries. The interpretation of a specific disease by dd-cfDNA should be combined with other clinical indicators.

Urinary donor-derived cell-free DNA as a non-invasive biomarker for BK polyomavirus-associated nephropathy.

BK polyomavirus-associated nephropathy (BKPyVAN) is a common cause of allograft failure. However, differentiation between BKPyVAN and type I T cell-mediated rejection (TCMR) is challenging when simian virus 40 (SV40) staining is negative, because of the similarities in histopathology. This study investigated whether donor-derived cell-free DNA (ddcfDNA) can be used to differentiate BKPyVAN. Target region capture sequencing was applied to detect the ddcfDNAs of 12 recipients with stable graft function, 22 with type I TCMR, 21 with proven BKPyVAN, and 5 with possible PyVAN. We found that urinary ddcfDNA levels were upregulated in recipients with graft injury, whereas plasma ddcfDNA levels were comparable for all groups. The median urinary concentrations and fractions of ddcfDNA in proven BKPyVAN recipients were significantly higher than those in type I TCMR recipients (10.4 vs. 6.1 ng/mL, P<0.001 and 68.4% vs. 55.3%, P=0.013, respectively). Urinary ddcfDNA fractions (not concentrations) were higher in the BKPyVAN-pure subgroup than in the BKPyVAN-rejection-like subgroup (81.30% vs. 56.64%, P=0.025). With a cut-off value of 7.81 ng/mL, urinary ddcfDNA concentrations distinguished proven BKPyVAN from type I TCMR (area under the curve (AUC)=0.848, 95% confidence interval (95% CI): 0.734 to 0.963). These findings suggest that urinary ddcfDNA is a non-invasive biomarker which can reliably differentiate BKPyVAN from type I TCMR.

Donor-derived cell-free DNA: An independent biomarker in kidney transplant patients with antibody-mediated rejection

IntroductionAntibody-mediated rejection (ABMR) is a major cause of kidney transplant failure which requires donor-specific antibodies (DSA) for a definitive diagnosis. Donor-derived cell-free DNA (ddcfDNA) is an emerging biomarker used to assess kidney allograft injury. However, current data is limited to predict the accuracy of ddcfDNA in ABMR diagnosis. This study was conducted to compare the performance of DSA with plasma ddcfDNA for the diagnosis of ABMR. MethodsIn this retrospective single-center observational study, we enrolled 50 kidney transplant recipients who were diagnosed with the suspicion of rejection between June 2018 and May 2019 at the Jinling Hospital. Plasma ddcfDNA was measured by using a novel target region capture sequencing methodology. A total of 37 patients who were tested with DSA and biopsy were divided into four subgroups (ABMR+/DSA+, ABMR+/DSA-, ABMR-/DSA+, ABMR-/DSA-) for the distribution of ddcfDNA (%) by ABMR and DSA. ResultsThe median level of ddcfDNA in biopsy showed that the ABMR group (1.66%, IQR 1.34–3.76%) was significantly higher than the median level (0.63%, IQR 0.43–0.74%) in non-ABMR (p < 0.001). With a ddcfDNA cutoff of 0.96%, the AUC was 0.90 (95%CI, 0.86–0.95), which was associated with a sensitivity of 90.5% (95%CI, 69.6–98.8%) and specificity of 96.6% (95%CI, 82.2–100%), a PPV of 95% (95%CI, 73.4–99.2%) and NPV of 93.3% (95%CI, 78.9–98.1%) were also observed. Among the four subgroups, ddcfDNA had no significant difference in both DSA+ group and DSA-group (p > 0.05). In the diagnosis of ABMR, the specificity, sensitivity, PPV and NPV of DSA were 50%, 74.1%, 41.7%, 80%, respectively. ConclusionsddcfDNA levels in the blood could highly distinguish (biopsy-supported) ABMR occurrence, irrespective of whether this method is accompanied by DSA or not.

Obesity treatment effect in Danish children and adolescents carrying Melanocortin-4 Receptor mutations

ObjectivesTo determine the prevalence of Melanocortin-4 Receptor (MC4R) mutations in a cohort of children and adolescents with overweight or obesity and to determine whether treatment responses differed between carriers and noncarriers.MethodsUsing target region capture sequencing, an MC4R mutation screen was performed in 1261 Danish children and adolescents enrolled at a tertiary multidisciplinary childhood obesity treatment center. Measurements of anthropometrics, blood pressure, fasting blood biochemistry including lipid and hormone levels, and dual-energy X-ray absorptiometry were performed at baseline and throughout treatment.ResultsOf 1209 children and adolescents that met all criteria to be included in the described analyses, 30 (2.5%) carried damaging or unresolved MC4R mutations. At baseline, mutation carriers exhibited higher concentrations of plasma thyroid-stimulating hormone (p = 0.003), and lower concentrations of plasma thyroxine (p = 0.010) compared to noncarriers. After a median of 1 year of treatment (range 0.5–4.0 years), body mass index (BMI) standard deviation score (SDS) was reduced in noncarriers but not in carriers, and this difference in treatment response was statistically significant (p = 0.005). Furthermore, HDL cholesterol was reduced in carriers, a response significantly different from that of noncarriers (p = 0.017).ConclusionAmong Danish children and adolescents with overweight or obesity entering a tertiary lifestyle intervention, 2.5% carried damaging or unresolved MC4R mutations. In contrast to noncarriers, carriers of damaging or unresolved MC4R mutations failed to reduce their BMI SDS during obesity treatment, indicating a need for personalized treatment based on the MC4R genotype.

Urine Donor-Derived Cell-Free DNA Helps Discriminate BK Polyomavirus-Associated Nephropathy in Kidney Transplant Recipients With BK Polyomavirus Infection.

Background: Studies have shown that plasma donor–derived cell-free DNA (dd-cfDNA) can predict renal allograft antibody-mediated rejection. This study was performed to evaluate the value of urine dd-cfDNA concentration and dd-cfDNA fraction (%) for discriminating BK polyomavirus-associated nephropathy (BKPyVAN) in kidney transplant recipients with urinary BK polyomavirus (BKPyV) infection.Methods: In this retrospective single-center observational study, we enrolled kidney transplant recipients who were diagnosed with urine BKPyV infection between August 2018 and May 2019 at the First Affiliated Hospital of Sun Yat-sen University. Urine dd-cfDNA was measured by using a novel target region capture sequencing methodology. The pathological diagnosis of BKPyVAN was confirmed by anti-SV40-T immunohistochemical staining and classified using the American Society for Transplantation schema. Receiver operating characteristic curve analysis was used to investigate the relations of urine dd-cfDNA and dd-cfDNA% to intrarenal allograft BKPyV infection states.Results: In total, 93 patients were enrolled, including 40 cases of proven BKPyVAN, seven cases of probable BKPyVAN, 23 cases of possible BKPyVAN, and 23 cases of resolving BKPyVAN. Urine dd-cfDNA level in proven BKPyVAN (22.09 ± 21.27 ng/ml) was comparable to that in probable BKPyVAN (15.64 ± 6.73 ng/ml, P = 0.434) but was significantly higher than that in possible BKPyVAN (5.60 ± 3.53 ng/ml) and resolving BKPyVAN (5.30 ± 3.34 ng/ml) (both Ps < 0.05). Urine dd-cfDNA% of proven BKPyVAN (0.71 ± 0.21) was lower than that of probable BKPyVAN (0.91 ± 0.04, P < 0.001), but was significantly higher than that of possible BKPyVAN (0.56 ± 0.30) and resolving BKPyVAN (0.46 ± 0.28) (both Ps < 0.05). For distinguishing biopsy-proven BKPyVAN from biopsy-excluded BKPyVAN, the discrimination capacity of urine dd-cfDNA (AUC: 0.842, 95% CI: 0.735, 0.918) was superior to that of plasma BKPyV DNA load (AUC: 0.660, 95% CI: 0.537, 0.769) with 0.181 (95% CI: 0.043, 0.319) difference between areas under ROC curves (P = 0.010).Conclusion: The elevated urine dd-cfDNA level may help discriminate BKPyVAN in kidney transplant recipients with BKPyV viruria.

Clinical and genomic features of high tumor mutation burden in patients with non-small cell lung cancer.

e21023 Background: TMB is associated with mono-immunotherapy efficacy for patients with advanced or metastatic non-small-cell lung cancer (NSCLC). For early-stage NSCLC or EGFR mutated non-resectable NSCLC, TMB-H predicted better prognosis or poor response to TKIs. This research might develop potential predictive parameters for distinguishing NSCLC with high or low TMB. Methods: Samples of surgically resected, cancer tissues were collected from 499 patients with NSCLC: 29 (5.8%) lung squamous cell carcinoma (LUSC) and 468 (94.2%) lung adenocarcinoma (LUAD). The mutations and TMB were confirmed by target region capture sequencing (Oseq™-508). Results: According to the median value (3.08), TMB is divided into high (N = 244) and low (N = 255). Interactions between TMB and sex, age, location, histology, pathological subtype, lymph node, parabronchial lymph node, LVI, neuro-invasive, stage and mutation status were evaluated. The distribution of TMB-H was significantly (p &lt; 0.001) correlated with sex, age, histology, pathological subtype, stage (TN stage) and partial mutations. Among all mutations, 6 genes (TP53 / FAT3 / KMT2D / TSHZ3 / NAV3 / EPHA3) were confirmed to be significantly related to TMB-H in NSCLC. We also found that TMB-H was significantly affected by mutation status of KRAS gene (P. G12X, p &lt; 0.001) and missense mutations in FAT3 (p &lt; 0.001). The Kruskal–Wallis H test results showed that the mutation types of FAT3, KRAS, TP53 and PIK3CA were closely related to TMB-H. Lasso linear regression analysis was applied and resulted the better predictors of TMB status including TSHZ3, KMT2D, TP53, gender and T stage. Conclusions: Instead of comprehensive genomic profiling to evaluate TMB, clinical characteristics and special mutation types may help to effectively screen and predicate patients with TMB-H status. [Table: see text]

Patho-genomic feature of PI3K / AKT / mTOR pathway mutations in Chinese patients with non-small cell lung cancer.

e21049 Background: Inhibition of PI3K/AKT/mTOR pathway has emerged as a promising anticancer strategy. Measuring genetic alterations in PI3K/AKT/mTOR pathway are critical for clinical decision making for patients with lung cancer. This study aims to analyze PI3K/AKT/mTOR pathway-related gene mutations in non-small cell lung cancer (NSCLC) in a consecutive cohort. Methods: 536 surgically resected tumor tissues were collected. Target region capture sequencing for 508 cancer-related genes was conducted on MGI-seq 2000 platform. Results: PI3K/AKT/mTOR pathway genes were mutated in 120 samples (22.4%). Female or older patients displayed higher mutation incidence of PI3K/AKT/mTOR pathway (P = 0.06 for female, P = 0.05 for patients ≥60 y). In NSCLC samples, mutations mainly occurred in NF1/2(18/499), PTEN (7/499), MTOR (4/499), TSC1/2(18/499), PIK3CA (26/499), PDK1 (2/499), AKT1/2/3 (5/499) and KRAS (49/499) genes. Genetic alterations of AKT2 (p = 0.003), mTOR (P = 0.03), PTEN (P = 0.005) and PIK3CA (P &lt; 0.001) are preferred to occur in squamous NSCLC. PIK3CA variants (p.E542K, p.E545K, p.Q546K) were more enriched in non-squamous NSCLC, while amplification of PIK3CA was more prevalent in squamous NSCLC (P = 0.038). Histologically, TSC1/2 mutations were more correlate with micro-invasion subtype in non-squamous NSCLC (P = 0.002). Meanwhile, PIK3CA alterations, especially, amplification occurred more often in keratinizing squamous NSCLC (P &lt; 0.001). In addition, four patients with biallelic NF1 loss of function mutations were exclusively found in non-squamous NSCLC without co-occurrence of mutation in known driver genes like EGFR or TP53, which indicated biallelic loss of function of NF1 might be sufficient to drive oncogenesis of non-squamous NSCLC. Conclusions: PI3K/AKT/mTOR pathway alterations present a heterogenous distribution across various types of lung malignancies especially in NSCLC. As more small molecule inhibitors developed to target PI3K/AKT/mTOR pathway, this study might shed light on further clinical investigation of these lately developed therapeutic approaches. [Table: see text]

Diverse phenotypes in children with PAX2-related disorder.

BackgroundThe aim of this study was to analyze the diverse phenotypes of children with PAX2‐related disorder so as to improve our understanding of this disease.MethodsThe clinical data of ten children with PAX2 mutations, detected by targeted region capture sequencing or whole‐exome sequencing, were retrospectively analyzed. Family members of index cases were verified by Sanger sequencing and family segregation analysis was performed.ResultsThe age of first symptom of 10 unrelated children (six girls and four boys) was 6.4 (ranged from postnatal day to 14.8) years old. Proteinuria, abnormal renal function, and structure were found in all patients. Renal hypoplasia and renal cysts were found in 10 of 10 and five of 10 cases, respectively. Three patients progressed to chronic kidney disease stage 5 and the onset age of end‐stage renal disease was 9.8–16.4 years old. PAX2‐related ocular abnormalities were found in five of seven cases and three patients were observed to have more than one ocular findings involved. In addition to diverse renal and ocular findings, new phenotypes including congenital ventricular septal defect, skeletal deformity (fourth metatarsal microsomia), ovarian teratoma, and relatively rare extrarenal manifestations such as growth retardation, gout, and microcephaly were also found. Three novel mutations were reported for the first time. De novo mutations occurred in all patients who were carried out segregation analysis. Patients with the same mutation had different manifestations. PAX2‐related disorder showed remarkable clinical variability and phenotypic heterogeneity.ConclusionWe firstly reported skeletal deformity (fourth metatarsal microsomia), ovarian teratoma, and congenital ventricular septal defect as new phenotypes of PAX2‐related disorder which enlarged the phenotypic spectrum. Gout was firstly reported as the onset symptom of PAX2‐related disorder. The diagnosis of PAX2‐related disorder should be considered without family history due to a much higher percentage of De novo mutations.

Genotype and clinical phenotype analysis in patients with retinitis pigmentosa and cone rod dystrophy

Objective To observe the gene mutation and clinical phenotype of patients with retinitis pigmentosa (RP) and cone rod dystrophy (CORD). Methods Thirty-seven patients with RP and 6 patients with CORD and 95 family members were enrolled in the study. The patient’s medical history and family history were collected. All the patients and family members received complete ophthalmic examinations to determine the phenotype, including best corrected visual acuity, slit lamp microscope, indirect ophthalmoscopy, color fundus photography, optical coherence tomography, full-field electroretinogram, and fluorescein fundus angiography. DNA was abstracted from patients and family members. Using target region capture sequencing combined with next-generation sequencing to screen the 232 candidate pathogenic mutations. Polymerase chain reaction and direct sequencing were used to confirm the pathogenic pathogenic mutations and Co-segregation is performed among members in the family to determine pathogenic mutation sites. The relationship between genotype and clinical phenotype of RP and CORD was analyzed. Results Of the 37 patients with RP, 13 were from 6 families, including 4 families with autosomal dominant inheritance, 2 families with autosomal recessive inheritance, and 3 in 6 families were detected pathogenic gene mutations. 24 cases were scattered RP. Six patients with CORD were from four families, all of which were autosomal recessive. Of the 43 patients, 21 patients were detected the pathogenic gene mutation, and the positive rate was 48.8%. Among them, 15 patients with RP were detected 10 pathogenic gene mutations including USH2A, RP1, MYO7A, C8orf37, RPGR, SNRNP200, CRX, PRPF31, C2orf71, IMPDH1, and the clinical phenotype included 10 typical RP, 2 cases of RPSP, 3 cases of Usher syndrome type 2 and 6 cases of CORD patients were all detected pathogenic gene mutations, including 2 cases of ABCA4, 2 mutations of RIMS1 gene, 1 case of CLN3 gene mutation, and 1 case of CRB1 and RPGR double gene mutation. Conclusions RP and CORD are clinically diverse in genotype and clinically phenotypically similar. For patients with early RP and CORD, clinical phenotype combined with genetic analysis is required to determine the diagnosis of RP and CORD. Key words: Retinal diseases/genetics; Retinitis pigmentosa/genetics; DNA mutational analysis; Phenotype

Dynamics of early post-operative plasma ddcfDNA levels in kidney transplantation: a single-center pilot study.

Donor-derived cell-free DNA (ddcfDNA) is reported to be a promising noninvasive biomarker for acute rejection in organ transplant. However, studies on monitoring ddcfDNA dynamics during the early periods after organ transplantation are scarce. Our study assessed the dynamic variation in ddcfDNA in early period with various types and status of kidney transplantation. Target region capture sequencing used identifies ddcfDNA level in 21 kidney transplant recipients. Median ddcfDNA level was 20.69% at the initial time post-transplant, and decreased to 5.22% on the first day and stayed at the stable level after the second day. The ddcfDNA level in DCD (deceased donors) group (44.99%) was significantly higher than that in LDRT (living donor) group (10.24%) at initial time, P<0.01. DdcfDNA level in DGF (delayed graft function) recipients was lower (23.96%) than that in non-DGF (47.74%) at the initial time, P=0.89 (19.34% in DGF and 4.46% in non-DGF on the first day, P=0.17). DdcfDNA level at initial time significantly correlated with serum creatinine (r2 =0.219, P=0.032) and warm ischemia time (r2 =0.204, P=0.040). Plasma ddcfDNA level decreased rapidly follow an L-shaped curve post-transplant, and level in DGF declined slower than non-DGF. The rebound of ddcfDNA level may indicate the occurrence of acute rejection.

A novel nonsense mutation in MYO15A is associated with non-syndromic hearing loss: a case report

BackgroundHearing loss is genetically heterogeneous and is one of the most common human defects. Here we screened the underlying mutations that caused autosomal recessive non-syndromic hearing loss in a Chinese family.Case presentationThe proband with profound hearing loss had received audiometric assessments. We performed target region capture and next generation sequencing of 127 known deafness-related genes because the individual tested negative for hotspot variants in the GJB2, GJB3, SLC26A4, and MTRNR1 genes. We identified a novel c.6892C > T (p.R2298*) nonsense mutation and a c.10251_10253delCTT (p.F3420del) deletion in MYO15A. Sanger sequencing confirmed that both mutations were co-segregated with hearing loss in this family and were absent in 200 ethnically matched controls. Bioinformatics analysis and protein modeling indicated the deleterious effects of both mutations. The p.R2298* mutation leads to a truncated protein and a loss of the functional domains.ConclusionsOur results demonstrated that the hearing loss in this case was caused by novel, compound heterozygous mutations in MYO15A. The p.R2298* mutation in MYO15A was reported for the first time, which has implications for genetic counseling and provides insight into the functional roles of MYO15A mutations.

Analysis of disease-causing gene mutation in three Chinese families with congenital inherited cataract

To identify the disease-causing gene mutations in three Chinese pedigrees affected with congenital inherited cataract, in ordre to provide genetic counseling and prenatal diagnosis. Using exons combined target region capture sequencing chip to screen the candidate disease-causing mutations, Sanger sequencing was used to confirm the disease-causing mutations. Family 1 was polymorphic cataract, family 2 was cerulean cataract, family 3 was coralliform cataract. The inheritance mode of the three pedigrees consisted with autosomal dominant inheritance. In family 1, a nonsense mutation of CRYβB2 gene c.463C>T in exon 6 result in a p.Q155X amino acid change. In family 2, a missense mutation of of CRYGD gene c.43C>T in exon 2 result in a p.R14C amino acid change. In family 3, a missense mutation of CRYGD gene c.70C>A in exon 2 result in a p.P23T amino aid change. No above-mentioned mutations were found in normal individuals. The nonsense mutation c.463C>T (p.Q155X) of CRYβB2 gene, the heterozygous mutations c.43C>T(p.R14C) of CRYGD gene and c.70C>A( p.P23T) of CRYGD gene was the disease-causing gene mutation in family 1, 2 and 3 respectively, our results provid genetic counseling and prenatal diagnosis for these three families.

Study of a family with epidermolysis bullosa simplex resulting from a novel mutation of KRT14 gene

To determine the molecular etiology for a Chinese pedigree affected with epidermolysis bullosa simplex (EBS). Target region sequencing using a hereditary epidermolysis bullosa capture array combined with Sanger sequencing and bioinformatics analysis were used. Mutation taster, PolyPhen-2, Provean, and SIFT software and NCBI online were employed to assess the pathogenicity and conservation of detected mutations. One hundred healthy unrelated individuals were used as controls. Target region sequencing showed that the proband has carried a unreported heterozygous c.1234A>G (p.Ile412Val) mutation of the KRT14 gene, which was confirmed by Sanger sequencing in other 8 affected individuals but not among healthy members of the pedigree. Bioinformatics analysis indicated that the mutation is highly pathogenic. Remarkably, 3 members of the family (2 affected and 1 unaffected) have carried a heterozygous c.1237G>A (p.Ala413Thr) mutation of the KRT14 gene, which was collected in Human Gene Mutation Database (HGMD). Bioinformatics analysis indicated that the mutation may not be pathogenic. Both mutations were not detected among the 100 healthy controls. The novel c.1234A>G(p.Ile412Val) mutation of the KRT14 gene is probably responsible for the disease, while c.1237G>A (p.Ala413Thr) mutation of KRT14 gene may be a polymorphism. Compared with Sanger sequencing, target region capture sequencing is more efficient and can significantly reduce the cost of genetic testing for EBS.

Identification of a novel missense mutation of MIP in a Chinese family with congenital cataracts by target region capture sequencing

Congenital cataract is both clinically diverse and genetically heterogeneous. To investigate the underlying genetic defect in three-generations of a Chinese family with autosomal dominant congenital cataracts, we recruited family members who underwent comprehensive ophthalmic examinations. A heterozygous missense mutation c.634G > C (p.G212R) substitution was identified in the MIP gene through target region capture sequencing. The prediction results of PolyPhen-2 and SIFT indicated that this mutation was likely to damage the structure and function of MIP. Confocal microscopy images showed that the intensity of the green fluorescent signal revealed much weaker signal from the mutant compared to the wild-type MIP. The expressed G212R-MIP was diminished and almost exclusively cytoplasmic in the HeLa cells; whereas the WT-MIP was stable dispersed throughout the cytoplasm, and it appeared to be in the membrane structure. Western blot analysis indicated that the protein expression level of the mutant form of MIP was remarkably reduced compared with that of the wild type, however, the mRNA levels of the wild-type and mutant cells were comparable. In conclusion, our study presented genetic and functional evidence for a novel MIP mutation of G212R, which leads to congenital progressive cortical punctate with or without Y suture.

Analysis of mutations of disease-causing gene and the inheritance in sporadic retinitis pigmentosa

Objective To observe the disease-causing genes and the inheritance in sporadic retinitis pigmentosa (sRP) in Ningxia region. Methods 49 sRP patients and 128 family members were recruited for this study. All the patients and family members received complete ophthalmic examinations including best corrected visual acuity, slit-lamp microscope, indirect ophthalmoscopy, fundus color photography, visual field, optic coherence tomography, full view electroretinogram. DNA was extracted from patients and family members. Using exon combined target region capture sequencing chip to screen the 230 candidate disease-causing gene mutations, polymerase chain reaction and direct sequencing were used to confirm the disease-causing mutations. Results 24/49 patients (49.0%) had identified disease-causing genes, totally 16 genes were involved. There were 41 mutation sites were found, including 32 new mutations (78.0%). The disease-causing genes include USH2A, C2orf71, GNGA1, RPGR1, IFT140, TULP1, CLRN1, RPE65, ABCA4, GUCA1, EYS, CYP4V2, GPR98 and ATXN7. Based on pedigree analysis, 20 patients were autosomal recessive retinitis pigmentosa, 3 patients were autosomal dominant retinitis pigmentosa and 1 patient was X linked retinitis pigmentosa. 3/7 patients with USH2A mutations were identified as Usher syndrome. Conclusions USHZA is the main disease-causing of sRP patients in Ningxia region. 83.3% of sRP in this cohort are autosomal recessive retinitis pigmentosa. Key words: Retinitis pigmentosa/genetics; Inheritance patterns; Genes; Mutation; High-throughput nucleotide sequencing

Low-coverage whole-genome sequencing and target-region capture sequencing in fetal congenital heart disease

Objective To explore the relationship between genetic phenotypes and clinical characteristics of fetal congenital heart disease(CHD) using low-coverage whole-genome sequencing and target sequencing. Methods Tissue specimens of 62 fetuses with CHD diagnosed by fetal echocardiography and confirmed by post-mortem autopsy from September 2012 to June 2015 were selected from the fetal cardiac malformation database of the Consultation Center of Maternal-Fetal Medicine in Fetal Heart Diseases, Beijing Anzhen Hospital, and classified according to the sectional embryology classification method. Low-coverage whole-genome sequencing and target sequencing were performed to detect chromosomal deletions or duplications and single nucleotide variation and small insertion/deletion fragment of CHD related genes. Results (1) Conotruncal defects (CTD) were the most common (69.4%, 43/62) seen CHD in this group. (2) Copy number variations (CNVs >100 kb) were detected in 30 fetuses and CNVs (>1 Mb) were detected in 11 fetuses out of the 30 fetuses (including seven fetuses with determined pathogenic CNVs, six of whom were involved with CTD). (3) Known or suspected pathogenic genes were detected in five fetuses, four of them had ventricular outflow tract obstruction. Conclusions CTD is most common in fetal CHD; most of the fetuses with pathogenic CNVs are complicated with CTD; and most of the fetuses with likely pathogenic genetic mutation are involved with ventricular outflow tract obstruction. Key words: Heart defects, congenital; Genome, human; Chromosome deletion; Chromosome duplication; Polymorphism, single nucleotide; Sequence analysis, DNA

A three-step programmed method for the identification of causative gene mutations of maturity onset diabetes of the young (MODY)

To establish a three-step programmed method to find gene mutations related to maturity onset diabetes of the young (MODY). Target region capture and next-generation sequencing (NGS) were performed using customized oligonucleotide probes designed to capture suspected genes for MODY in 11 probands with clinically diagnosed MODY. The suspected associations of certain genes with MODY were then confirmed by Sanger sequencing in the probands and their family members. Finally, to validate variants of one of the genes of interest (glucokinase, GCK) as pathogenic mutations, protein function editing by the variant genes was assessed. In the target region capture and NGS phase, a total of nine variants of seven genes (GCK, WFS1, SLC19A2, SH2B1, SERPINB4, RFX6, and GATA6) were identified in eight probands. Two heterozygous GCK mutations located on the same allele (p.Leu77Arg and p.Val101Met) were identified in a MODY family. Sanger sequencing was used to confirm the variants identified by NGS to be present in probands and their diabetic family members, but not in non-diabetic family members. Finally, enzyme kinetic and thermal stability analyses revealed that the p.Leu77Arg mutation or the p.Leu77Arg mutation in combination with the p.Val101Met mutation inactivates GCK function and stability, while mutation of p.Val101Met alone does not. The p.Leu77Arg but not p.Val101Met GCK mutation is therefore considered a pathogenic mutation associated with MODY. Genetic screening coupled with gene-editing protein function testing is an effective and reliable method by which causative gene mutations of MODY can be identified.

A Novel Nonsense Mutation of POU4F3 Gene Causes Autosomal Dominant Hearing Loss.

POU4F3 gene encodes a transcription factor which plays an essential role in the maturation and maintenance of hair cells in cochlea and vestibular system. Several mutations of POU4F3 have been reported to cause autosomal dominant nonsyndromic hearing loss in recent years. In this study, we describe a pathogenic nonsense mutation located in POU4F3 in a four-generation Chinese family. Target region capture sequencing was performed to search for the candidate mutations from 81 genes related to nonsyndromic hearing loss in this family. A novel nonsense mutation of POU4F3, c.337C>T (p. Gln113⁎), was identified in a Chinese family characterized by late-onset progressive nonsyndromic hearing loss. The novel mutation cosegregated with hearing loss in this family and was absent in 200 ethnicity-matched controls. The mutation led to a stop codon and thus a truncated protein with no functional domains remained. Transient transfection and immunofluorescence assay revealed that the subcellular localization of the truncated protein differed markedly from normal protein, which could be the underlying reason for complete loss of its normal function. Here, we report the first nonsense mutation of POU4F3 associated with progressive hearing loss and explored the possible underlying mechanism. Routine examination of POU4F3 is necessary for the genetic diagnosis of hereditary hearing loss in the future.

Analysis of gene mutation in a Chinese family with low penetrance retinoblastoma

Objective To identify the pathogenic mutation in a three generation Chinese family with low penetrance retinoblastoma (RB). Methods 8 from 9 family members received complete ophthalmic examinations. DNA was extracted from 6 family members. Using exon combined target region capture sequencing chip to screen the candidate disease-causing mutations. Sanger sequencing were used to confirm the disease-causing mutation. Results Among 9 family members, the proband (Ⅲ2) was bilateral RB, Ⅲ1 was unilateral RB, Ⅲ3 was dead for bilateral RB. Normal fundus were observed in the left eye of Ⅲ1 and the eyes of other family members except the proband. Sequence analysis of RB1 gene revealed a missense mutation c. 1981C>T (p.Arg661Trp) in the proband and two carriers (Ⅱ2, Ⅱ3), but not in the two normal subjects (Ⅱ1, Ⅱ4). We suspect that the RB penetrance in the family was 50%. Conclusions There is a missense mutation c. 1981C>T in a Chinese family with low penetrance RB. The RB penetrance is 50%. Key words: Retinoblastoma; Genes, neoplasm; Penetrance; High-throughput nucleotide sequencing