[A case of tuberous sclerosis complex due to a novel splicing variant of TSC2 gene].

OBJECTIVE: To explore the genetic basis for a patient with tuberous sclerosis complex. METHODS: Genomic DNA was extracted from peripheral blood samples from members of his family and 100 unrelated healthy controls. The proband was subjected to next-generation sequencing, and candidate variant was confirmed by multiple ligation-dependent probe amplification (MLPA) and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was carried out to determine the relative mRNA expression in the proband. RESULTS: The patient was found to harbor a c.2355+1G>C splicing variant of the TSC2 gene. Sequencing of cDNA confirmed that 62 bases have been inserted into the 3' end of exon 21, which has caused a frameshift producing a truncated protein. CONCLUSION: The novel splicing variant c.2355+1G>C of the TSC2 gene probably underlay the TSC in the proband. Above finding has expanded the variant spectrum of TSC2 and provided a basis for preimplantation genetic testing and/or prenatal diagnosis.

Complex preimplantation genetic tests for Robertsonian translocation, HLA, and X-linked hyper IgM syndrome caused by a novel mutation of CD40LG gene.

PURPOSE: To perform complex preimplantation genetic tests (PGT) for aneuploidy screening, Robertsonian translocation, HLA-matching, and X-linked hyper IgM syndrome (XHIGM) caused by a novel mutation c.156 G>T of CD40LG gene. METHODS: Reverse transcription PCR (RT-PCR) and Sanger sequencing were carried out to confirm the causative variant of CD40LG gene in the proband and parents. Day 5 and D6 blastocysts, obtained by in vitro fertilization (IVF) with intracytoplasmic sperm injection, underwent trophectoderm (TE) biopsy and whole genomic amplification (WGA) and next generation sequencing (NGS)-based PGT to detect the presence of a maternal CD40LG mutation, aneuploidy, Robertsonian translocation carrier, and human leukocyte antigen (HLA) haplotype. RESULTS: Sanger sequencing data of the genomic DNA showed that the proband has a hemizygous variant of c. 156 G>T in the CD40LG gene, while his mother has a heterozygous variant at the same position. Complementary DNA (cDNA) of CD40LG amplification and sequencing displayed that no cDNA of CD40LG was found in proband, while only wild-type cDNA of CD40LG was amplified in the mother. PGT results showed that only one of the six tested embryos is free of the variant c.156 G>T and aneuploidy and having the consistent HLA type as the proband. Meanwhile, the embryo is a Robertsonian translocation carrier. The embryo was transplanted into the mother's uterus. Amniotic fluid testing results are consistent with that of PGT. A healthy baby girl was delivered, and the peripheral blood testing data was also consistent with the testing results of transplanted embryo. CONCLUSIONS: The novel mutation of c. 156 G>T in CD40LG gene probably leads to XHIGM by nonsense-meditated mRNA decay (NMD), and complex PGT of preimplantation genetic testing for monogenic disease (PGT-M), aneuploidy (PGT-A), structural rearrangement (PGT-SR), and HLA-matching (PGT-HLA) can be performed in pedigree with both X-linked hyper IgM syndrome and Robertsonian translocation.

[Novel mutations of XPC gene detected in a family affected with xeroderma pigmentosum group C].

OBJECTIVE: To detect mutations of the XPC (XPC complex subunit, DNA damage recognition and repair factor) gene in a family affected with xeroderma pigmentosum group C (XP-C). METHODS: The patient was subjected to next-generation sequencing and Sanger sequencing. Suspected mutations were validated by Sanger sequencing. Effect of splicing mutation was confirmed by reverse transcription-PCR (RT-PCR). RESULTS: Compound heterozygous mutations of c.2098G to T and c.2034-7_2040del were found in the XPC gene in the proband. Among these, c.2098G to T (p.G700X) is a nonsense mutation resulting in a truncated XPC protein. C.2034-7_2040del involves the -1 position, which may alter the splice donor site of the intron 11 of XPC and result in a truncated XPC protein with loss of amino acids from 940 to 679 positions. The two mutations were not detected among 100 unrelated healthy controls. CONCLUSION: Mutations of c.2098 G to T and c.2034-7_2040del of the XPC gene may lead to abnormal XPC expression and reduction or elimination of normal XPC functions, which may underlie the disease in this family.

[A novel mutation of GLI3 gene underlying synpolydactyly in a family].

OBJECTIVE: To detect mutation of GLI3 gene in a family affected with autosomal dominant synpolydactyly. METHODS: Genomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated healthy controls. Potential mutation was screened by next-generation sequencing and confirmed by Sanger sequencing. RESULTS: A heterozygous frameshift mutation c.480dupC was identified in the GLI3 gene among all patients from the family. The same mutation was not found in unaffected family members and the 100 healthy controls. CONCLUSION: The c.480dupC of the GLI3 gene probably underlies the synpolydactyly in this family.

Reproductive management through integration of PGD and MPS-based noninvasive prenatal screening/diagnosis for a family with GJB2-associated hearing impairment.

A couple with a proband child of GJB2 (encoding the gap junction protein connexin 26)-associated hearing impairment and a previous pregnancy miscarriage sought for a reproductive solution to bear a healthy child. Our study aimed to develop a customized preconception-to-neonate care trajectory to fulfill this clinical demand by integrating preimplantation genetic diagnosis (PGD), noninvasive prenatal testing (NIPT), and noninvasive prenatal diagnosis (NIPD) into the strategy. Auditory and genetic diagnosis of the proband child was carried out to identify the disease causative mutations. The couple then received in-vitro-fertilization treatment, and eight embryos were obtained for day 5 biopsy. PGD was performed by short-tandem-repeat linkage analysis and Sanger sequencing of GJB2 gene. Transfer of a GJB2c.235delC heterozygous embryo resulted in a singleton pregnancy. At the 13th week of gestation, genomic DNA (gDNA) from the trio family and cell-free DNA (cfDNA) from maternal plasma were obtained for assessment of fetal chromosomal aneuploidy and GJB2 mutations. NIPT and NIPD showed the absence of chromosomal aneuploidy and GJB2-associated disease in the fetus, which was later confirmed by invasive procedures and postnatal genetic/auditory diagnosis. This strategy successfully prevented the transmission of hearing impairment in the newborn, thus providing a valuable experience in reproductive management of similar cases and potentially other monogenic disorders.

Novel missense mutation in WNT6 in 100 couples with unexplained recurrent miscarriage.

STUDY QUESTION: Do mutations and/or polymorphisms in coding sequences in Wingless-Type MMTV Integration Site Family, Member 6 (WNT6) play a role in unexplained recurrent miscarriage (unexplained RM) in Chinese couples? SUMMARY ANSWER: We found four mutations in the coding sequences of WNT6 which appear to exist in a small proportion of Chinese women with unexplained RM. WHAT IS KNOWN ALREADY: WNT6 has been proved to be essential for stromal cell proliferation during decidualization in mice, but in humans WNT6 has not been studied in recurrent miscarriage populations. STUDY DESIGN, SIZE, DURATION: For this study, 100 couples with unexplained RM (at least three or more unexplained spontaneous miscarriages), and 100 ethnically matched fertile couples (at least one live birth and no history of pregnancy pathologies) were recruited. All the participants were chosen over a 7-year period from the National Research Center for Assisted Reproductive Technology and Reproductive Genetics at Shandong University, Jinan, China. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were recruited following extensive clinical studies. Genomic DNA was isolated from peripheral blood. Mutation analysis in the coding regions of WNT6 was performed by PCR amplification and DNA sequences testing in all participants. Functional effects of missense variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT). MAIN RESULTS AND THE ROLE OF CHANCE: Four rare novel mutations, including one missense mutation, were found in intron 1, exon 3 and the 3' untranslated region of WNT6 in four women with unexplained RM. Gene software predictions showed that the missense mutation in exon 3 could alter the function of WNT6. No mutations or polymorphisms were detected in the male partners of the unexplained RM patients or in the fertile controls. To further validate the findings, we continued to screen this missense mutation site in another 100 peripheral blood samples of normal fertile females, and there was still no positive result. LIMITATIONS, REASONS FOR CAUTION: There is no direct evidence to validate whether these novel mutations discovered in the present research are related to unexplained RM. Further studies are warranted to investigate the role of WNT6 in unexplained RM, including larger studies in an independent group. WIDER IMPLICATIONS OF THE FINDINGS: These results provide evidence to suggest the importance of WNT6 in reproductive failure and may support the hypothesis that WNT6 is essential for stromal cell proliferation during decidualization. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Science and Technology Development Planning of Shandong (2013GGE27001), the National Natural Science Foundation of China (81300459), the Science Projection of Bureau of Public Health in Weifang (2012044) and the Science Research Foundation Item of No-earnings Health Vocation. The authors have no competing interests to declare.

Human fetal trophonema matrix and uterine endometrium support better human embryonic stem cell growth and neural differentiation than mouse embryonic fibroblasts.

The concerns over xenogeneic pathogens and immunogenic molecules derived from mouse embryonic fibroblasts (MEFs) trigger the development of human-derived feeder layers for human embryonic stem cell (hESC) maintenance. It is essential to evaluate the capability of these human feeder layers to retain the stemness and pluripotency of hESCs. In the present study, two Chinese hESC lines, SDU-hESCm-1 and SDU-hESCm-2, were continuously cultured on human adult uterine endometrial cells (hUEC), human fetal trophonema matrix cells (hFTMC), and MEFs for at least two month (up to 10 passages). A side-by-side comparison of the abilities to support: (1) self-renewal of the hESCs, (2) expression of undifferentiated markers, and (3) neural differentiation, was made between the human and mouse feeder layers. We demonstrated that the hESCs maintained on hUEC and hFTMC exhibited significantly higher growth rates and generated higher levels of DNA content than those on MEFs. Under neural differentiation-promoting conditions, greater neural differentiation was found in the hESCs maintained on human than on mouse feeder layers. These results suggest that human feeder layers derived from hUECs and hFTMCs are more efficient in supporting a long-term growth and neural differentiation of hESCs than MEFs.