Identification of a novel TBX5 c.755 + 1 G > A variant and related pathogenesis in a family with Holt-Oram syndrome.

The proband with congenital heart disease and abnormal thumb was clinically diagnosed as Holt-Oram syndrome (HOS). A novel variant, T-box transcription factor 5 (TBX5) c.755 + 1 G > A, was identified in the proband via whole exome sequencing and validated using Sanger sequencing. Pedigree analysis and clinical examinations revealed three/seven individuals over three generations within the family, with features suggestive of HOS. Deep amplicon sequencing confirmed that the allele frequencies of the novel variant in the proband (III-1), her brother (III-2), and her mother (II-2) were 50%, 48.3%, and 38.1%, respectively, indicating that III-1 and III-2 harbored heterozygous variants, while II-2 harbored mosaic heterozygous variants. The minigene splicing assay showed that the novel variant affected the normal splicing of exon 7, resulting in the production of abnormal TBX5 transcripts. Reverse transcription-quantitative polymerase chain reaction and western blot analyses revealed that the novel variant upregulated TBX5 expression at the transcriptional and translational levels. Nuclear localization assay demonstrated impaired nuclear localization of the mutant TBX5. Cell viability assay revealed the inhibition of cell activity by the mutant TBX5. Our findings indicate that the novel variant was potentially induced HOS, probably by causing aberrant splicing, reducing the enrichment of nuclear TBX5 protein, and inhibiting cellular proliferation.

Elevated Prevalence of Abnormal Glucose Metabolism and Other Endocrine Disorders in Patients with ß-Thalassemia Major: A Meta-Analysis.

BACKGROUND: Endocrinopathies are common in patients with ß-thalassemia major despite parenteral iron chelation therapy with deferoxamine. Prevalence of abnormal glucose metabolism in previous studies was controversial. The aim of this study was to discuss the prevalence of abnormal glucose metabolism in ß-thalassemia major based on a meta-analysis. METHODS: PubMed, ScienceDirect, Springerlink, Ovid, Web of Science, MEDLINE, Wanfang database, and Chinese National Knowledge Internet were searched for relevant articles. Two authors selected the articles according to the inclusion criteria and then extracted the data. The prevalence of diabetes mellitus (DM) in ß-thalassemia major was defined as the primary outcome. The prevalence with the 95% confidence interval (95%CI) was used to evaluate the proportion of abnormal glucose metabolism and other endocrine disorders in patients with ß-thalassemia major. Subgroup analyses were applied to explore the prevalence in different regions. Sensitivity analysis and publication bias assessment were also conducted. RESULTS: A total of 44 studies with 16605 cases were included in this analysis. Diabetes mellitus was present in 6.54% (95% CI: 5.30%-7.78%). The fixed subgroup study revealed that the region with the highest prevalence was the Middle East (prevalence= 7.90%, 95% CI: 5.75%-10.05%). The accumulated meta-analysis revealed that the prevalence of DM in ß-thalassemia major was relatively steady in each year. The prevalence of impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and other endocrine disorders in ß-thalassemia major was 17.21% (95% CI: 8.43%-26.00%), 12.46% (95% CI: 5.98%-18.94%), and 43.92% (95% CI: 37.94%-49.89%), respectively. Sensitivity analysis showed that the pooled results were robust; publication bias assessment revealed that there was no significant evidence that the pooled results were influenced by publication bias. CONCLUSION: High prevalence of endocrine disorders involving abnormal glucose metabolism was detected in ß-thalassemia major. Treatment and prevention measurements may be necessary to prevent growth and endocrine problems.

Chromosomal aneuploidies and copy number variations in posterior fossa abnormalities diagnosed by prenatal ultrasonography.

OBJECTIVE: To explore the genetic aetiology of fetal posterior fossa abnormalities (PFAs). METHODS: This study involved cases of PFAs that were identified by prenatal ultrasonographic screening and confirmed postnatally between January 2012 and January 2016. Conventional cytogenetic analyses and chromosomal microarray analysis were performed, and chromosomal aneuploidies and copy number variations (CNVs) were identified. RESULTS: Among 74 cases included in this study, 8 were of Blake's pouch cyst; 7, Dandy-Walker malformation; 11, vermian hypoplasia; 32, enlarged cisterna magna; and 16, cerebellar hypoplasia. The rates of nonbenign chromosomal aberrations (including chromosomal aneuploidies, pathogenic CNVs, and variants of unknown significance) were 2/8 (25.0%), 2/7 (28.5%), 8/11 (72.7%), 7/32 (21.9%), and 6/16 (37.5%), respectively. Cases were also classified as isolated PFAs (30/74), PFAs with other central nervous system (CNS) abnormalities (13/74), or PFAs with extra-CNS structural abnormalities (31/74). No fetuses with isolated PFAs or PFAs accompanied by other CNS abnormalities exhibited chromosomal aneuploidies or pathogenic CNVs. The rate of pathogenic chromosomal aberrations in the remaining fetuses was 17/31 (22.9%). CONCLUSION: The combined use of chromosomal microarray analysis and karyotype analysis might assist the prenatal diagnosis and management of PFAs, with extra-CNS structural abnormalities being detected by ultrasonography.

Prenatal Diagnosis of a Case of Norrie Disease with Late Development of Bilateral Ocular Malformation.

We report a case of Norrie disease, diagnosed by prenatal ultrasound, confirmed by Sanger sequencing of the DNP gene from the aborted fetal cord blood and histologically. Prenatal ultrasound revealed no abnormality in either eye at 22+1 and 31+4 gestational weeks, but at 36+5 gestational weeks both eyes had massive vitreous cavity opacities with complete retinal detachment. Norrie disease was initially suspected because of an older male sibling with the disease. To our knowledge, prenatal ultrasound diagnosis of Norrie disease has been previously described only one case in 1993 in a 34-week-old fetus. The normal eye development until after 31 + 4 gestational weeks provides insight into the first manifestation and then the rapid progression of the eye disease.

Prenatal diagnosis of submicroscopic chromosomal aberrations in fetuses with ventricular septal defects by chromosomal microarray-based analysis.

OBJECTIVES: To evaluate the usefulness of chromosomal microarray analysis in fetuses with ventricular septal defects (VSDs) with or without associated anomalies and normal karyotype. METHODS: Fetuses with VSDs and normal karyotypes were investigated by using an Affymetrix CytoScan HD array. The cases were classified as isolated or nonisolated VSDs. RESULTS: Among the 52 VSD fetuses, 22 (42.3%) had isolated defects and 30 (57.7%) had additional other ultrasound anomalies. Twenty-six CNVs were identified in 18 fetuses (34.6%), 15 benign CNVs were detected in 11 (21.2%) fetuses, and 8 pathogenic CNVs were detected in 6 (11.5%) fetuses. After excluding 2 fetuses with 22q11.2 deletion syndrome, the rate of pathogenic CNVs was 7.7%. The proportion of variants of unknown significance was 5.8% (3/52). In five cases, additional malformations were detected after birth or abortion, and one case had a prenatal isolated VSD. The detection rate of pathogenic CNVs in nonisolated VSDs was nonsignificantly higher than that in prenatal or postnatal isolated VSDs (4.5%, 1/22 vs 16.7%, 5/30, P = 0.226; 0/21 vs 19.4%, 6/31, P = 0.07). CONCLUSIONS: The results demonstrated the value of chromosomal microarray analysis in the prenatal diagnosis of VSDs. The complexity of other defects enhanced the frequency of pathogenic CNVs, although the results were not significantly different. © 2016 John Wiley & Sons, Ltd.

MCDA twins with discordant malformations: submicroscopic chromosomal anomalies detected by chromosomal microarray analysis and clinical outcomes.

OBJECTIVE: To evaluate whether discrepant copy number variations (CNVs) contribute to the risk for discordant congenital anomalies in monochorionic diamniotic (MCDA) twins. METHODS: We conducted a parallel testing using both G-banding for standard karyotyping and chromosomal microarray analysis (CMA) with Affymetrix CytoScan HD array in MCDA twins with discordant malformations. RESULTS: During the study period, 193 MCDA twins with discordant malformations were detected and followed up. Multiple anomalies and cardiac defects were detected most frequently among the fetuses with malformations. Among all the 119 MCDA twins that were successfully performed fetal karyotyping, discordance of chromosomal aberrations were identified in nine cases, including one with discordant trisomy 18, seven with discordant monosomy X, one twin with 47, XXY and the co-twin with 45, X [7]/46, XY[43]. CMA revealed pathological CNVs in four out of the 110 fetuses with normal karyotype and the detection rate of uncertain clinical significance was 3.6% (4/110). Discordance of CNVs was detected in 5.5% (3/55) among the 55 MCDA twins with normal karyotype. Monozygosity was confirmed in all the 61 MCDA twins that were performed CMA. CONCLUSIONS: Large whole chromosome abnormalities are more common between discordant twins rather than smaller CNVs in this study. © 2016 John Wiley & Sons, Ltd.

Improved assay performance of single nucleotide polymorphism array over conventional karyotyping in analyzing products of conception.

BACKGROUND: Conventional karyotyping has been a routine method to identify chromosome abnormalities in products of conception. However, this process is being transformed by single nucleotide polymorphism (SNP) array, which has advantages over karyotyping, including higher resolution and dispensing with cell culture. Therefore, the purpose of this study was to evaluate the advantage of high-resolution SNP array in identifying genetic aberrations in products of conception. METHODS: We consecutively collected 155 products of conception specimens, including 139 from first-trimester miscarriage and 16 from second-trimester miscarriage. SNP array was performed on these samples in parallel with G-banded karyotyping. RESULTS: The test success rate was 98.1% (152/155) using SNP array, which was higher than that using karyotyping (133/155, 85.8%). It yielded a 63.8% (97/152) abnormality rate, and the frequency of various chromosome abnormalities was in agreement with other previous studies. The results between array and karyotyping demonstrated a 94.0% (125/133) concordance. SNP array obtained additional aberrations in 3.8% (5/133) of those cases unidentified by karyotyping, which included three cases with whole-genome uniparental disomy, one with pathogenic copy number variation, and one with del(4)(q35.1q35.2) and dup(12)(q24.31q24.33). However, chromosome translocations presented in two cases and tetraploidy presented in one case were detected by karyotyping instead of array. Additionally, two out of three cases with mosaic trisomy were revealed by array but recognized as pure trisomy by karyotyping. CONCLUSION: This study demonstrated that SNP array had certain advantages over G-banded karyotyping, including a higher success rate, additional detection of copy number variations and uniparental disomy, and improved sensitivity to mosaicism. Therefore, it would be an alternative method to karyotyping in clinical genetic practice.

[Investigation of ultrasound markers in screening fetal trisomy 21].

OBJECTIVE: To investigate the clinical value of ultrasound markers in screening fetal trisomy 21. METHODS: From Jan. 2001 to Dec. 2011, a retrospective study about sonographic information of 138 fetuses diagnosed as trisomy 21 was taken in the First Affiliated Hospital of Sun Yat-sen University. All fetuses were divided into 3 groups: isolated ultrasound markers, non-isolated ultrasound markers, and isolated structural malformations or other abnormalities. The relationship between trisomy 21 and ultrasound markers as well as structural anomalies or other abnormalities was analyzed. RESULTS: Sonographic anomalies were detected in 132 fetuses (95.7%, 132/138), including ultrasound markers and structural malformations or other abnormalities. One hundred and twenty cases (87.0%, 120/138) had ultrasound markers, 38 (31.7%, 38/120) had one marker and 82 (68.3%, 82/120) had more than one marker (P < 0.01). Fifty-one fetuses (37.0%, 51/138) had isolated ultrasound markers and non-isolated markers were found in 69 fetuses (50.0%, 69/138). Only 12 fetuses (8.7%, 12/138) had isolated structural malformations or other abnormalities. In 20 fetuses on whom the first-trimester ultrasound screening were performed, all had ultrasound markers, 95% (19/20) had thickened nuchal translucency and 55% (11/20) had nasal bone hypoplasia. The most common ultrasound markers on the second-trimester screening were nasal bone hypoplasia, which accounted for 41.9% (52/124) cases, followed by thickened nuchal fold (25.0%, 31/124), short femurs and humerus (24.2%, 30/124), echogenic intracardiac focus (16.1%, 20/124), mild ventriculomegaly (15.3%, 19/124), hyperechoic bowel (12.9%, 16/124), mild renal pyelectasis (12.1%, 15/124). Furthermore, the common structural malformations or other abnormalities were as follows: cardiac defects (33.1%, 41/124), digestive system (26.6%, 33/124). CONCLUSIONS: Ultrasound markers are valuable for screening fetal trisomy 21. The fetuses of trisomy 21 usually had more than one ultrasound markers or associated with other abnormalities. Combinations of ultrasound markers with the results of serum screening and maternal age are necessary for evaluation.

[Spectral karyotyping of seven prenatally detected marker chromosomes and complex chromosome aberrations].

OBJECTIVE: To perform spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) and conventional karyotyping on prenatally detected marker chromosomes and complex chromosomal aberrations. METHODS: Five marker chromosomes and 2 complex chromosome aberrations diagnosed by G banding were collected. SKY was performed to verify the composition of marker chromosomes. FISH was used to confirm the diagnosis when necessary. In certain cases, C or N banding technique was employed to verify the composition of chromosomes. Results of ultrasonography and pregnancy outcome were reviewed. RESULTS: Among the 5 marker chromosomes, 2 were large and 3 were medium in size, 4 were de novo and one was inherited from the father. By SKY analysis, 2 marker chromosomes have originated from non-acrocentric chromosomes (4 and 9), whilst the other two have originated from acrocentric chromosomes (21 and 22). The remainder was derived from X chromosome. The SKY results were confirmed by FISH in 3 cases. Four cases have chosen to terminate the pregnancy after genetic counseling. A fetus with inherited paternal marker chromosome was delivered at term, and showed normal development during the first year of life. As for the other 2 cases with complex chromosome aberrations, by SKY examination, one had duplication in chromosome 8 and the other had chromosome rearrangements derived from translocation between chromosomes 2 and 6. In the latter case the fetus was delivered at term but showed developmental retardation at 6 months. CONCLUSION: SKY in combination with FISH can facilitate identification of the origins of marker chromosomes as well as complex chromosomal aberrations. With combined information from ultrasonography, SKY and FISH, effective counseling may be offered to the patients.

[Clinical features of abnormal chromosome karyotypes in twin pregnancies complicated with structural abnormalities].

OBJECTIVE: To investigate the clinical features of the abnormal chromosome karyotypes in twin pregnancies complicated with fetal malformations. METHODS: Totally 181 twin pregnancies (362 fetuses) in which one or two fetuses had abnormalities diagnosed by ultrasound were referred to the First Affiliated Hospital of Sun Yat-Sen University from January, 2000 to September, 2010. They were divided into different groups according to (1) maternal age: the cases with maternal age ≥ 35 were divided into advanced pregnancy group (105 fetuses), and those with maternal age < 35 were divided into young pregnancy group (203 fetuses); (2) conceived method: those conceived by assisted reproductive technology were divided into assisted reproductive group (81 fetuses), and the natural conception pregnancies were divided into natural conception group (227 fetuses); (3) chorionicity: the monochorionic twin (MCT) pregnancies were divided into MCT group (123 fetuses), and the dichorionic twin (DCT) pregnancies were divided into DCT group (185 fetuses); (4) structural abnormalities: 205 fetuses with structural abnormalities were divided into the abnormal fetal group, and 103 fetuses without structural abnormalities were divided into the normal fetal group. All fetuses were examined by the ultrasound and chromosomes were examined in 308 fetuses. RESULTS: (1) The karyotype of fetuses: among 181 twin pregnancies, 23 cases had chromosomal abnormalities in 1 or 2 fetuses (12.7%, 23/181), and chromosomes were examined in both fetuses in 20 of 23 cases. Twenty-six of 308 fetuses were found with abnormal chromosomes (8.4%, 26/308), and the aneuploid was the most common type of abnormal karyotypes (53.8%, 14/26). Twenty-one of 205 fetuses with malformations were found with abnormal karyotypes (10.2%, 21/205). (2) Seven of 123 fetuses in MCT group were with abnormal karyotypes (5.7%, 7/123), and 19 of 185 fetuses in DCT group were with abnormal karyotypes (10.3%, 19/185). There was no statistical difference of abnormal chromosome incidence between the two groups. There were 14 fetuses with aneuploid in DCT group (7.6%, 14/185); but there was no fetus with aneuploid in MCT group. There was statistical difference between these two groups. In two cases of DCT group, only one fetus with malformation received chromosome examination because another fetus was dead, and the karyotypes were trisomy 21 and trisomy 18 respectively. Both fetuses of the rest 17 cases received chromosome examination, and the chromosomes of both fetuses in each pregnancy were different. Fifteen of 19 fetuses with abnormal chromosomes in DCT group were complicated with structural abnormalities, and 7 fetuses of 4 twin pregnancies in MCT group were with chromosomal abnormalities. (3) The comparison of the abnormal karyotype incidence between the advanced pregnancy group and young pregnancy group: the abnormal karyotype incidence of the advanced pregnancy group was 7.6% (8/105), and that was 8.9% (18/203) in young pregnancy group. There was no statistical difference between the two groups (P > 0.05). Six of 105 fetuses in advanced pregnancy group were aneuploids (5.7%, 6/105), and 8 of 203 fetuses in young pregnancy group were aneuploids (3.9%, 8/203). The aneuploid incidence in advanced pregnancy group was significantly higher than that in young pregnancy group (P < 0.05).(4) The comparison of the abnormal karyotype incidence between the assisted reproductive group and the natural conception group: 11 of 81 fetuses were with the abnormal karyotypes in assisted reproductive group (13.6%, 11/81), and 15 of 227 fetuses were with the abnormal karyotypes in assisted reproductive group (6.6%, 15/227). There was statistical difference between the two groups (P < 0.05). There were 7 fetuses with the aneuploid in assisted reproductive group (8.6%, 7/81) and 7 fetuses with the aneuploid in natural conception group (3.1%, 7/227), which showed no statistical difference (P > 0.05). (5) The comparison of the abnormal karyotype incidence between the abnormal fetal group and normal fetal group: 21 of 205 fetuses in abnormal fetal group were with abnormal karyotypes (10.2%, 21/205), and 5 of 103 fetuses in normal fetal group were with abnormal karyotypes (4.9%, 5/103). There was no statistical difference (P > 0.05). 13 fetuses in abnormal fetal group were with the aneuploid (6.3%, 13/205), and only one fetus in normal fetal group was aneuploid (1.0%, 1/103). There was statistical difference between the two groups (P < 0.05). CONCLUSIONS: Aneuploid is the most common abnormal karyotype in twin pregnancy complicated with fetal abnormalities, especially trisomy 21. Aneuploid mainly occurs in only one fetus of DCT, and chromosomal discordance is usually found in DCT. While in MCT, the twin fetuses with the same abnormal karyotype may have different phenotypes. The results suggest that it is necessary to analyze both karyotypes of twins even if only one fetus is complicated with structural abnormalities.

[Combined use of molecular cytogenetic techniques to detect a small chromosomal translocation].

OBJECTIVE: Comprehensive use of molecular cytogenetic techniques for the detection of 1 case of small chromosome translocation. METHODS: Following conventional chromosome preparation, G-banding karyotype analysis, spectral karyotyping (SKY), whole chromosome painting, two-color fluorescence in situ hybridization (FISH) and subtelomeric probe FISH were performed. RESULTS: G-banded karyotype was 46, XX, ?(22q11.3), SKY karyotype analysis was 46, XX, der (4)t(4;6) and found no abnormalities on chromosome 22, staining signal was not found with any abnormalities on chromosome 6. Two-color FISH indicated a chromosomal translocation segment of 22q13.3 to one end of the short arm of chromosome 4. Subtelomeric FISH probe showed the end of the long arm of chromosome 22 and the end of the short arm of chromosome 4 reciprocal translocation. High resolution G-banding and FISH result indicated 46, XX, t(4;22)(p15.3;q13.2). CONCLUSION: The testing of small chromosomal translocation should be combined with clinical information and integrated use of molecular cytogenetic techniques to improve the accuracy of diagnosis of chromosomal diseases.

[Middle segment pancreatectomy for the benign tumors of the neck and body of the pancreas (report of 15 cases)].

OBJECTIVE: To study the clinical application value of middle segment pancreatectomy in the treatment of benign tumors of the amphi-neck of the pancreas. METHODS: Fifteen cases were retrospectively analyzed treated from November 2005 to June 2009. There were 3 male and 12 female aging from 30 to 50 years. They all received middle segment pancreatectomy for benign tumors of the amphi-neck of the pancreas. RESULTS: There was no death during perioperative period. All the 15 patients received middle segment pancreatectomy. Fourteen of them received the closure of broken ends of pancreatic head, pancreaticojejunostomy (mono-anastomosis) and the rest one received dipl-anastomosis. Postoperative pathology showed that in the 15 patients, 1 got solid-pseudopapillary tumor of the pancreas, 3 got non-functional islet cell tumor, 11 got cystadenoma of pancreas. Three of them got pancreatic fistula and were self cured in 3 months. Follow-up visits to all the patients kept in the following 2 to 45 months. There was no death. No patients got new-onset diabetes and pancreatic pseudocyst. And their tumors were not relapsed. CONCLUSIONS: There is an exact therapeutic effect of middle segment pancreatectomy for benign tumors of the amphi-neck of the pancreas. The treatment has little function damage to patients' endocrine and external secretion. The incidence rate of pancreatic fistula in middle segment pancreatectomy is higher than that in pancreaticoduodenectomy. As long as the drainage is kept unobstructed, most of the pancreatic fistula can be self cured.

[Gene therapy targeting for carcinoma regulated by E2F-1 promoter].

OBJECTIVE: To study the selective effect to tumor cells mediated by a recombinant adenoviral vector carrying E2F-1 promoter. METHODS: The AdEasy-1 adenoviral vector system was used in this experiment. Several recombinant adenovirus with tumor-targeting E2F-1 promoter were constructed and then the E2F-1 promoter gene was checked by PCR and sequencing. The two adenovirus expressing GFP gene which is regulated by E2F-1 promoter or CMV promoter were used to respectively transfect tumor cells and non-proliferating normal cells, then observed and analyzed the different results caused by different promoters. Vpr gene was cloned into the targeting recombinant adenovirus. The new adenovirus named rvAdE2F-1/vpr was used to transfect tumor cells SMMC-7721, LS174T and non-proliferating normal cells H292, L-02. The surviving rate of each group was registered; the level of E2F-1 protein expressed in normal and tumor cell lines were checked by Western Blot. RESULT: E2F-1 promoter can regulate the downstream gene GFP selectively expressed in LS174T and its activity in LS174T was similar with CMV promoter's; Vpr gene regulated by E2F-1 promoter can suppress the proliferation of tumor cells and no toxicity to normal cells; In all of the tumor cells, a much higher level of E2F-1 was expressed compared with normal cell lines. E2F-1 promoter's activity correlated well with E2F-1 protein levels. CONCLUSIONS: E2F-1 promoter can control a selective cell killing to cancer cells, with no effect to normal cells. The system of E2F-1 promoter is a useful method for tumor-targeting gene therapy.