An assessment of the analytical performance of non-invasive prenatal testing (NIPT) in detecting sex chromosome aneuploidies: 34,717-patient sample in a single prenatal diagnosis Centre in China.

OBJECTIVE: The present study aimed to evaluate the efficacy of a non-invasive prenatal test (NIPT) in the detection of the sex chromosome aneuploidies (SCAs) at our prenatal diagnosis centre. METHODS: Among a cohort of 34,717 pregnancies, maternal plasma samples from our prenatal diagnosis centre were subject to analysis of SCAs using NIPT detection. Pregnant women with NIPT positive results of SCAs were recommended to undergo an invasive prenatal diagnosis (i.e. karyotyping and fluorescence in situ hybridization) to validate the prediction value of NIPT. RESULTS: From 34,717 clinical pregnancies, 229 (0.66%) pregnancies were identified with SCAs. Of these, 78 (34.1%) cases were positive for 45,X and 151 (65.9%) cases comprised a sex chromosome trisomy. Of the 229 positive NIPT results, 193 (84.3%) cases had accepted an invasive diagnosis involving karyotyping analysis of the amniotic fluid, which confirmed 67 cases (34.7%) as true positive, as well as 126 cases (65.3%) as false positive. The positive predictive values were 23.07%, 50%, 36% and 27.27% respectively. The remaining 36 (15.7%) cases declined a prenatal diagnosis. The termination rates of 45,X, 47,XXY, 47,XXX and 47,XYY were 20.5%,46%,12.9% and 11.5% respectively. CONCLUSIONS: NIPT demonstrated a lower accuracy in predicting monosomy X than sex chromosome trisomies. After invasive testing, the fetal chromosome with 45,X and 47,XXY were terminated more often than those with 47,XXX, 47,XYY. Because NIPT is a screening test, false positive/negative cases exist, and pre- and post-test counselling is essential for informing patients about the benefits and limitations of the test. Confirmatory testing of abnormal results is recommended prenatally or after birth, and the importance of confirmatory testing and benefits of early diagnosis should be addressed.

[Factors affecting the failure of non-invasive prenatal testing and the feasibility analysis of retesting].

OBJECTIVE: To explore the cause for the failure of non-invasive prenatal testing (NIPT) and feasibility of repeated testing. METHODS: Clinical data, test results and pregnancy outcomes of 40 311 pregnant women who received NIPT test from January 2011 to December 2018 were reviewed. RESULTS: Among all the pregnant women, 1116 cases failed in the first test, 9 cases (0.81%) had fetal free DNA concentration lower than 4%, 663 cases (59.41%) were retested after the establishment of Z value gray area, and the remainder 444 cases (39.78%) needed to be retested after the blood collection due to the fetal free DNA concentration lower than 4%. After retesting, 1069 cases (95.78%) obtained effective NIPT results. The results showed that 53 cases were at high risk (6 cases for trisomy 21, 6 cases for trisomy 18, 13 cases for trisomy 13, 16 cases for sex chromosomal abnormality, 12 cases for chromosomal copy number variation). Forty-eight cases were selected for invasive prenatal diagnosis, and 2 cases of 47, XXY and 2 CNV were confirmed. A total of 47 cases (0.12%) did not obtain results because the concentration of fetal free DNA was lower than 4%. Only 16 cases (34%) chose invasive prenatal diagnosis. CONCLUSION: Repeated detection of the gray area of Z value can reduce the false positive rate of NIPT and invasive prenatal diagnosis, and the feasibility of repeated detection is high. In the case of fetal free DNA concentration lower than 4%, the success rate of obtaining effective NIPT results by re-sampling and re-detection increases with the increase of gestational age, but may delay the diagnosis for fetal aneuploidies. Therefore, personalized estimation should be made according to gestational age and clinical indications. It is suggested that pregnant women should choose invasive prenatal diagnosis when they have failed in the retest.

Spermatogenic phenotype of testis-specific protein, Y-encoded, 1 (TSPY1) dosage deficiency is independent of variations in TSPY-like 1 (TSPYL1) and TSPY-like 5 (TSPYL5): a case-control study in a Han Chinese population.

Testis-specific protein, Y-encoded, 1 (TSPY1) is involved in the regulation of spermatogenic efficiency via highly variable copy dosage, with dosage deficiency of the multicopy gene conferring an increased risk of spermatogenic failure. TSPY-like 1 (TSPYL1) and TSPY-like 5 (TSPYL5), two autosomal homologous genes originating from TSPY1, share a core sequence that encodes a functional nucleosome assembly protein (NAP) domain with TSPY1. To explore the potential effects of TSPYL1 and TSPYL5 on the TSPY1-related spermatogenic phenotype, we investigated the expression of these genes in 15 healthy and nonpathological human tissues (brain, kidney, liver, pancreas, thymus, prostate, spleen, muscle, leucocytes, placenta, intestine, ovary, lung, colon and testis) and explored associations between their variations and spermatogenic failure in 1558 Han Chinese men with different spermatogenic conditions, including 304 men with TSPY1 dosage deficiency. TSPYL1 and TSPYL5 were expressed in many different tissues, including the testis. An unreported rare variant that is likely pathogenic (c.1057A>G, p.Thr353Ala) and another of uncertain significance (c.1258C>T, p.Arg420Cys) in the NAP-coding sequence of TSPYL1 were observed in three spermatogenesis-impaired patients with heterozygous status. The distribution differences in the alleles, genotypes and haplotypes of eight TSPYL1- and TSPYL5-linked common variants did not reach statistical significance in comparisons of patients with spermatogenic failure and controls with normozoospermia. No difference in sperm production was observed among men with different genotypes of the variants. Similar results were obtained in men with TSPY1 dosage deficiencies. Although the distribution of missense variants of TSPYL1 found in the present and other studies suggests that patients with spermatogenic failure may have a statistically significant greater burden of rare variations in TSPYL1 relative to normozoospermic controls, the functional evidence suggests that TSPYL1 contributes to impaired spermatogenesis. Moreover, the present study suggests that the effects of TSPYL1 and TSPYL5 on the spermatogenic phenotype of TSPY1 dosage deficiency are limited, which may be due to the stability of their function resulting from high sequence conservation.

[Discussion on the standard of clinical genetic testing report and the consensus of gene testing industry].

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.

Evidence for the involvement of the proximal copy of the MAGEA9 gene in Xq28-linked CNV67 specific to spermatogenic failure.

Spermatogenic failure characterized by impaired sperm production is a common multifactorial disease with molecular and cytogenetic causes for its extreme phenotype that include azoospermia and severe oliogzoospermia. Recently, a high-resolution array-comparative genomic hybridization analysis of the X chromosome and a subsequent cohort study revealed three X-linked microdeletions (CNV64, CNV67, and CNV69) that were associated with decreased sperm production in a mixed group that included Spanish and Italian males. To confirm their spermatogenic effect, we examined the hemizygous deletions and copy dosage of the MAGE family member A9 (MAGEA9) gene, which is a potential X-linked candidate for the CNV67-related spermatogenic phenotype, to investigate their association with spermatogenic failure in 1722 Han males from southwest China. The individuals in this group consisted of 884 patients with idiopathic azoospermia/oliogzoospermia and 838 controls with normozoospermia. Our results showed that both CNV64 and CNV69 were more common in patients than in controls. Similar to that reported previously, the CNV67 was also identified as being specific to spermatogenic failure in our population, although it was rare. More importantly, the paralog ratio tests and sequence family variant analyses provided evidence that the CNV67 might cause a partial deletion of the proximal copy of the MAGEA9 and suggests that CNV67-related spermatogenic failure may be attributed to the functional defect of the Cancer/Testis gene. Our findings highlight the potential of the Xq-linked CNV67 to serve as a novel detection target in the etiological diagnosis of spermatogenic failure and male infertility, although its pathogenic mechanism remains to be elucidated.

Copy number variation of functional RBMY1 is associated with sperm motility: an azoospermia factor-linked candidate for asthenozoospermia.

STUDY QUESTION: What is the influence of copy number variation (CNV) in functional RNA binding motif protein Y-linked family 1 (RBMY1) on spermatogenic phenotypes? SUMMARY ANSWER: The RBMY1 functional copy dosage is positively correlated with sperm motility, and dosage insufficiency is an independent risk factor for asthenozoospermia. WHAT IS KNOWN ALREADY: RBMY1, a multi-copy gene expressed exclusively in the adult testis, is one of the most important candidates for male infertility in the azoospermia factor (AZF) region of the Y-chromosome. RBMY1 encodes an RNA-binding protein that serves as a pre-mRNA splicing regulator during spermatogenesis, and male mice deficient in Rbmy are sterile. STUDY DESIGN, SIZE, DURATION: A total of 3127 adult males were recruited from 2009 to 2016; of this group, the dosage of RBMY1 functional copy were investigated in 486 fertile males. In the remaining 2641 males with known spermatogenesis status, 1070 Y-chromosome haplogroup (Y-hg) O3* or O3e carriers without chromosomal aberration or known AZF structure mutations responsible for spermatogenic impairment, including 506 men with normozoospermia and 564 men with oligozoospermia or/and asthenozoospermia, were screened, and the RBMY1 functional copy dosage and copy conversion were determined to explore their associations with sperm phenotypes. The correlation between RBMY1 dosage and its mRNA level or RBMY1 protein level and the correlation between sperm RBMY1 level and motility were analysed in 15 testis tissue samples and eight semen samples. Ten additional semen samples were used to confirm the subcellular localization of RBMY1 in individual sperm. PARTICIPANTS/MATERIALS, SETTING, METHODS: All the Han volunteers donating whole blood, semen and testis tissue were from southwest China. RBMY1 copy number, copy conversion, mRNA/protein amount and protein location in sperm were detected using the AccuCopy® assay method, paralog ratio test, quantitative PCR, western blotting and immunofluorescence staining methods, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: This study identified Y-hg-independent CNV of functional RBMY1 in the enrolled population. A difference in the distribution of RBMY1 copy number was observed between the group with normal sperm motility and the group with asthenozoospermia. A positive correlation between the RBMY1 copy dosage and sperm motility was identified, and the males with fewer than six copies of RBMY1 showed an elevated risk for asthenozoospermia relative to those with six RBMY1 copies, the most common dosage in the population. The RBMY1 copy dosage was positively correlated with its mRNA and protein level in the testis. Sperm with high motility were found to carry more RBMY1 protein than those with relatively low motility. The RBMY1 protein was confirmed to predominantly localize in the neck and mid-piece region of sperm as well as the principal piece of the sperm tail. Our population study completes a chain of evidence suggesting that RBMY1 influences the susceptibility of males to asthenozoospermia by modulating sperm motility. LIMITATIONS REASONS FOR CAUTION: High sequence similarity between the RBMY1 functional copies and a large number of pseudogenes potentially reduces the accuracy of the copy number detection. The mechanism underlying the CNV in RBMY1 is still unclear, and the effect of the structural variations in the RBMY1 copy cluster on the copy dosage of other protein-coding genes located in the region cannot be excluded, which may potentially bias our observations. WIDER IMPLICATIONS OF THE FINDINGS: Asthenozoospermia is a multi-factor complex disease with a limited number of proven susceptibility genes. This study identified a novel genomic candidate independently contributing to the condition, enriching our understanding of the role of AZF-linked genes in male reproduction. Our finding provides insight into the physiological and pathological characteristics of RBMY1 in terms of sperm motility, supplies persuasive evidence of the significance of RBMY1 copy number analysis in the clinical counselling of male infertility resulting from asthenozoospermia. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the National Natural Science Foundation of China (Nos. 81370748 and 30971598). The authors have no conflicts of interest.

Primary azoospermia factor C duplication associated with spermatogenic impariment: a case-control study based on Y-chromosome haplogrouping in a Han Chinese population.

BACKGROUND: Azoospermia factor C (AZFc) in the male-specific region of Y-chromosome (MSY) presents wide structure variation mainly due to frequent non-allele homologous recombination, leading to significant copy number variation of the AZFc-linked coding sequences involving in spermatogenesis. A large number of studies had been conducted to investigate the association between AZFc deletions and male infertility in certain Y chromosome genetic backgrounds, however, the influence of primary AZFc duplication on spermatogenesis remained controversial and the cause of the discrepant outcomes is unknown. METHODS: In the present study, a total of 1,102 unrelated Han Chinese males without any detectable AZF deletions were recruited from 2014 to 2019, including 411 controls with normozoospermia and 691 patients with idiopathic spermatogenic failure. Using multiple paralog ratio tests (PRTs), the structure duplications were classified by the copy number of the AZFc-linked amplicons and genes. The Y-chromosome haplogroup (Y-hg) was categorized by genetyping of MSY-linked polymorphism loci. The association of primary AZFc duplication with spermatogenic phenotype was investigated in males with the same Y-hg. RESULTS: Within Y-hg O3* group, the frequency of the gr/gr duplication in patients is significantly higher than that of controls (P = 1.29×10-3 , odds ratio (OR) 7.64, 95% confidence interval (CI) 1.79-32.57). Moreover, Y-hg O3* males with the gr/gr duplication presented a significantly lower sperm production compared with non-AZFc duplicated ones (sperm concentration: P = 1.46×10-3 ; total sperm count: P = 1.82 ×10-3 ). The b2/b3 duplication were identified clustered in Y-hg Cα2*, and the significant difference in the distribution was not observed between patients with spermatogenic failure and controls. CONCLUSION: The results suggest that, in the Han Chinese population, the gr/gr duplication is a predisposing genetic factor for spermatogenic impairment in males harboring Y-hg O3* . Meanwhile, the b2/b3 duplication may be fixed on a yet-unidentified subbranch of Y-hg Cα2* without significantly deleterious effect on spermatogenesis. Our findings provide evidence that the difference in the Y-hg composition may cause the discrepancy on the association of AZFc duplication with spermatogenic failure among the studied populations.

BRD4L cooperates with MYC to block local tumor invasion via suppression of S100A10.

Targeted therapy based on BRD4 and MYC shows promise due to their well-researched oncogenic functions in cancer, but their tumor-suppressive roles are less understood. In this study, we employ a systematic approach to delete exons that encode the low-complexity domain (LCD) of BRD4L in cells by using CRISPR-Cas9. In particular, the deletion of exon 14 (BRD4-E14) results in cellular morphological changes towards spindle-shaped and loosely packed. BRD4-E14 deficient cells show increased cell migration and reduced cell adhesion. The expression of S100A10 was significantly increased in cells lacking E14. BRD4L binds with MYC via the E14-encoded region of the LCD to inhibit the expression of S100A10. In cancer tissues, there is a positive correlation between BRD4 and MYC, while both of these proteins are negatively associated with S100A10 expression. Finally, knocking out the BRD4-E14 region or MYC promotes tumor growth in vivo. Together, these data support a tumor-suppressive role of BRD4L and MYC in some contexts. This discovery emphasizes the significance of a discreetly design and precise patient recruitment in clinical trials that testing cancer therapy based BRD4 and MYC.

Combined analysis of genome-wide-linked susceptibility loci to Kawasaki disease in Han Chinese.

Kawasaki disease (KD) is a dominant cause of acquired heart disease in children due to frequent complicating coronary artery lesions (CALs). Genome-wide association study and linkage analysis have recently identified 6 susceptibility loci at genome-wide significance of P < 5.0 × 10(-8) in subjects of Japanese, Taiwanese and European. In present study, we analysed the variants of 6 single nucleotide polymorphisms (SNPs) in the genetic loci to investigate their potential effect on KD susceptibility and outcomes in Han Chinese population. As a result, the risk alleles of rs1801274 and rs2254546 were observed significant effect on KD with higher frequencies in 358 patients than those in 815 controls. The significant role of rs1801274, rs2857151 and rs2254546 in KD was found in the multi-variable logistic regression analysis of the SNPs. Two 2-locus and one 3-locus combinations of the SNPs showed significant effect on KD with stronger association with KD relative to comparable single SNP or 2-locus combinations. Significant susceptibility to CALs was found in KD patients with high-risk genotypes at both rs1801274 and rs2857151. The meta-analyses first revealed significant risk for CALs in KD patients carrying risk allele of rs11340705, and the association of rs28493229 with KD was not observed in the Han Chinese. In conclusion, the findings demonstrated that 5 of the 6 genetic loci influence the risk for KD and 3 of them may be involved in secondary CALs formation in Han Chinese. The additive effects of 3 multi-locus combinations on KD/CALs imply that some loci may participate together in certain unknown gene networks related to KD/CALs. Further function studies of the genetic loci are helpful for better understanding the pathophysiology of KD.

Association between the first and second trimester cell free DNA fetal fraction and spontaneous preterm birth.

OBJECTIVE: To evaluate whether the fetal fraction of cell-free DNA at the first and second trimesters is associated with spontaneous preterm birth. METHODS: This was a retrospective cohort study with singleton pregnancies who underwent noninvasive prenatal testing. According to pregnancy outcome, eligible patients were divided into a delivery group ≥37 weeks of pregnancy (term group) and <37 weeks of pregnancy (spontaneous preterm group). Stepwise linear regression was used to identify maternal characteristics associated with the fetal fraction of cell-free DNA. Logistic regression analysis was performed to evaluate the association between the fetal fraction of cell-free DNA and spontaneous preterm birth, adjusted for confounding factors. RESULTS: 14,020 cases were included in the study, 13292 cases (94.81%) in the term group and 728 cases (5.19%) in the spontaneous preterm group. The cell-free fraction of fetal DNA was inversely correlated with maternal age and body mass index. Positively correlated with gestational age, fertility, and assisted reproductive technology. After adjusting for the covariates, logistic regression analysis revealed no statistically significant association between the fetal fraction of cell-free DNA and spontaneous preterm birth. CONCLUSION: In our original study, we found no association between the fetal fraction on NIPT and subsequent spontaneous preterm birth.

Prenatal diagnosis identifies compound heterozygous variants in RYR1 that causes ultrasound abnormalities in a fetus.

OBJECTIVE: We presented a non-consanguineous healthy Chinese couple with five pregnancies, three early miscarriages, the fetus II-2 and II-5 with similar abnormal phenotypes of fetal hydrops, scoliosis, fetal akinesia and polyhydramnios. This study aimed to uncover the molecular etiology of this family with a history of multiple adverse pregnancies. MATERIALS AND METHODS: DNA extracted from the fifth fetal umbilical cord and parents' peripheral blood were subjected to SNP-array and whole exome sequencing. The result was verified by Sanger sequencing. Functional characterization of the c.2682G > C (p.Ile860_Pro894del) variant was completed by minigene splicing assay. RESULTS: Trio whole-exome sequencing has identified compound heterozygous variants in RYR1 (c.2682G > C; p.Ile860_Pro894del and c.12572G > A; p.Arg4191His) in fetus II-5. The variant c.2682G > C (p.Ile860_Pro894del) comes from the father and the c.12572G > A (p.Arg4191His) comes from the mother. The c.2682G > C (p.Ile860_Pro894del) affects the splice site resulting in exon 21 skipping, therefore is classified as likely pathogenic. The c.12572G > A (p.Arg4191His) locates in the C-terminal hot spots region of the RYR1, classified as of uncertain significance. CONCLUSIONS: We report the first prenatal case of RYR1-related disorders in Chinese population, expanding the variant spectrum of RYR1 in fetuses.

Case Report: Preimplantation Genetic Testing for Meckel Syndrome Induced by Novel Compound Heterozygous Mutations of MKS1.

Meckel syndrome (MKS), also known as the Meckel-Gruber syndrome, is a severe pleiotropic autosomal recessive developmental disorder caused by dysfunction of the primary cilia during early embryogenesis. The diagnostic criteria are based on clinical variability and genetic heterogeneity. Mutations in the MKS1 gene constitute approximately 7% of all MKS cases. Herein, we present a non-consanguineous couple with three abnormal pregnancies as the fetuses showed MKS-related phenotypes of the central nervous system malformation and postaxial polydactyly. Whole-exome sequencing identified two novel heterozygous mutations of MKS1: c.350C>A and c.1408-14A>G. The nonsense mutation c.350C>A produced a premature stop codon and induced the truncation of the MKS1 protein (p.S117*). Reverse-transcription polymerase chain reaction (RT-PCR) showed that c.1408-14A>G skipped exon 16 and encoded the mutant MKS1 p.E471Lfs*92. Functional studies showed that these two mutations disrupted the B9-C2 domain of the MKS1 protein and attenuated the interactions with B9D2, the essential component of the ciliary transition zone. The couple finally got a healthy baby through preimplantation genetic testing for monogenic disorder (PGT-M) with haplotype linkage analysis. Thus, this study expanded the mutation spectrum of MKS1 and elucidated the genetic heterogeneity of MKS1 in clinical cases.

Loss-of-function mutations in CEP78 cause male infertility in humans and mice.

Centrosomal protein dysfunction might cause ciliopathies. However, the role of centrosomal proteins in male infertility remains poorly defined. Here, we identified a pathogenic splicing mutation in CEP78 in male infertile patients with severely reduced sperm number and motility, and the typical multiple morphological abnormalities of the sperm flagella phenotype. We further created Cep78 knockout mice, which showed an extremely low sperm count, completely aberrant sperm morphology, and approximately null sperm motility. The infertility of the patients and knockout mice could not be rescued by an intracytoplasmic sperm injection treatment. Mechanistically, CEP78 might regulate USP16 expression, which further stabilizes Tektin levels via the ubiquitination pathway. Cep78 knockout mice also exhibited impairments in retina and outer hair cells of the cochlea. Collectively, our findings identified nonfunctional CEP78 as an indispensable factor contributing to male infertility and revealed a role for this gene in regulating retinal and outer hair cell function in mice.

CEP128 is involved in spermatogenesis in humans and mice.

Centrosomal proteins are necessary components of the centrosome, a conserved eukaryotic organelle essential to the reproductive process. However, few centrosomal proteins have been genetically linked to fertility. Herein we identify a homozygous missense variant of CEP128 (c.665 G > A [p.R222Q]) in two infertile males. Remarkably, male homozygous knock-in mice harboring the orthologous CEP128R222Q variant show anomalies in sperm morphology, count, and motility. Moreover, Cep128 knock-out mice manifest male infertility associated with disrupted sperm quality. We observe defective sperm flagella in both homozygous Cep128 KO and KI mice; the cilia development in other organs is normal-suggesting that CEP128 variants predominantly affected the ciliogenesis in the testes. Mechanistically, CEP128 is involved in male reproduction via regulating the expression of genes and/or the phosphorylation of TGF-ß/BMP-signalling members during spermatogenesis. Altogether, our findings unveil a crucial role for CEP128 in male fertility and provide important insights into the functions of centrosomal proteins in reproductive biology.

X Chromosome Inactivation Pattern and Pregnancy Outcome of Female Carriers of Pathogenic Heterozygous X-Linked Deletions.

Prenatal risk assessment of carriers of heterozygous X-linked deletion is a big challenge due to the phenotypic modification induced by X chromosome inactivation (XCI). Herein, we described four Chinese pedigrees with maternal-inherited X-deletions above 1 Mb. The pathogenic evaluation revealed that all X-deletions are harmful to heterozygous carriers; however, the asymptomatic pregnant female carriers in these families tremendously complicate the prognostic assessment of the unborn heterozygous embryos. In this study, we detected the XCI pattern of 11 female carriers of heterozygous X-linked deletions and 4 non-carrier females in these families and performed the first prenatal XCI pattern analysis in a fetal female carrier of heterozygous PCDH19-deletion to make risk prediction. In an adult female who lost one copy of the terminal of X chromosome short arm (Xp), a region enriching a large number of XCI escapees, the expression level of representative XCI escape genes was also detected. Pregnancy outcomes of all families were followed up or retrospected. Our research provides clinical evidence that X-deletions above 1 Mb are indeed associated with extremely skewed XCI. The favorable skewed XCI in combination with potential compensatory upregulation of XCI escapees would protect some but not all female carriers with pathogenic X-deletion from severe clinical consequences, mainly depending on the specific genetic contents involved in the deletion region. For PCDH19-disorder, the XCI pattern is considered as the decisive factor of phenotype expression, of which prenatal XCI assay using uncultured amniocytes could be a practicable way for risk prediction of this disease. These results provide valuable information about the usage of XCI assay in the prenatal risk assessment of heterozygous X-linked deletions.

Prenatal Diagnosis and Genetic Analysis of 21q21.1-q21.2 Aberrations in Seven Chinese Pedigrees.

Background: Chromosomal aberrations contribute to human phenotypic diversity and disease susceptibility, but it is difficult to assess their pathogenic effects in the clinic. Therefore, it is of great value to report new cases of chromosomal aberrations associated with normal phenotypes or clinical abnormalities. Methods: This was a retrospective analysis of seven pedigrees that carried 21q21.1-q21.2 aberrations. G-banding and single-nucleotide polymorphism array techniques were used to analyze chromosomal karyotypes and copy number variations in the fetuses and their family members. Results: All fetuses and their family members showed normal karyotypes in seven pedigrees. Here, it was revealed that six fetuses carried maternally inherited 21q21.1-q21.2 duplications, ranging from 1 to 2.7 Mb, but none of the mothers had an abnormal phenotype. In one fetus, an 8.7 Mb deletion of 21q21.1-q21.2 was found. An analysis of the pedigree showed that the deletion was also observed in the mother, brother, and maternal grandmother, but no abnormal phenotypes were found. Conclusion: This study identified 21q21.1-q21.2 aberrations in Chinese pedigrees. The carriers of 21q21.1-q21.2 duplications had no clinical consequences based on their phenotypes, and the 21q21.1-q21.2 deletion was transmitted through three generations of normal individuals. This provides benign clinical evidence for pathogenic assessment of 21q21.1-q21.2 duplication and deletion, which was considered a variant of uncertain significance and a likely pathogenic variant in previous reports.

Novel biallelic variants in COL7A1 cause recessive dystrophic epidermolysis bullosa.

BACKGROUND: Autosomal recessive dystrophic epidermolysis bullosa (RDEB) is an incurable and severe inherited skin disorder characterized by recurrent blistering at the sublamina densa beneath the cutaneous basement membrane. It is caused by biallelic loss-of-function mutation in the gene encoding type VII collagen (COL7A1). This study aimed to identify the causative variants of a Chinese RDEB patient and further provide prenatal diagnosis for the ongoing risk pregnancy of the proband's mother. METHODS: Clinical exome sequencing (CES) has been performed and an in-house pipeline was used to conduct a phenotype-driven data analysis. A minigene assay was used to verify the pathogenicity of a novel splice site variant in the COL7A1. RESULTS: Here we report two compound heterozygous variants in COL7A1, c.3867delT (p.G1290Efs*35) and c.5532+4_5532+5delAG, identified in a RDEB patient by CES. The minigene assay confirmed that thec.5532+4_5532+5delAGchange was a noncanonic splice site variant leading to in an in-frame deletion of exon 64. Prenatal diagnosis indicated that the present pregnancy of the patient's mother was not affected. CONCLUSION: Our study expands the mutation spectrum of COL7A1 and demonstrated that CES and minigene assays were efficient tools for RDEB molecular diagnoses.

Prenatal diagnosis of familial submicroscopic duplication at 18q22.3 without phenotypic abnormalities.

OBJECTIVE: To report a case of familial submicroscopic duplication at 18q22.3 without phenotypic abnormalities. CASE REPORT: Here, we reported two different cases with novel copy number variation at chromosome 18q22.3: one carried a maternally inherited 2.36 Mb microduplication, and the other carried a patrilineally inherited 1.74 Mb microduplication. The HumanCytoSNP-12 array allows for the visualization of the CNVs and maps the breakpoints. Both parents with the microduplication at 18q22.3 as well as their foetuses had normal phenotypes; the infants were regularly followed up after one year of age, and no abnormalities were found, including abnormalities related to growth, intelligence and sexual development. CONCLUSION: Our report showed that the duplication of 18q22.3 (chr18:68,606,012-71,287,101) might represent a benign variant.

A retrospective analysis the clinic data and follow-up of non-invasive prenatal test in detection of fetal chromosomal aneuploidy in more than 40,000 cases in a single prenatal diagnosis center.

OBJECTIVE: To evaluate the efficacy of non-invasive prenatal test (NIPT) in the detection of chromosomal aneuploidy according to the follow-up information from a single prenatal diagnosis center. METHODS: A total of 40,311 cases were retrospectively reviewed. The screening was performed using a BGI protocol, pre-test and post-test genetic counseling was provided, and the pregnancy outcomes were recorded. The results of NIPT and clinical follow-up data were analyzed together with the pregnancy outcomes, confirmatory testing results, and ultrasound findings. RESULTS: Of the 40,311cases were includes in the study, successful follow-up was conducted in 468 (1.16%) cases with high risk, 225 (0.56%) cases with rare autosomal trisomy (RAT) and copy number variation (CNV). 39,572 (98.17%) cases with low risk and 623 (1.57%) cases of which were confirmed with adverse pregnancy outcomes. 46 (0.1%) cases with failed tests. Among them, 398 (84.7%) cases with high-risk results chose invasive testing, revealing 198 true positive cases. In cases with RAT and CNV results, 189 cases underwent invasive testing, revealing 5 cases RAT and 4 pathogenic CNVs. CONCLUSIONS: NIPT appears to be effective in detecting the fetal chromosomal aneuploidies T21, T18 and SCAs, but it exist false positive/negative cases, unconfirmed high-risk cfDNA results, and the high false positive rate in cases with RAT and CNV results implied the limitations of this screening method. Our study showed the importance to associate cfDNA screening results with clinical follow-up data and provided information that may help with result interpretation, genetic counseling and the decision making in clinic.

Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization.

Preimplantation genetic screening (PGS) detects chromosomal aneuploidy from DNA extracted from trophectodermal biopsy of the embryos before implantation. Although a controlled study showed no difference in pregnancy rates between this invasive cell biopsy technique and a non-biopsied control group, the potential long-term damage by the current PGS method has not be completely ruled out. We therefore tested a less-invasive protocol which utilizes spent culture medium combining with blastocoel fluid (ECB) to assess chromosomal aneuploidy. We compared the new protocol with the currently employed trophectodermal biopsy method against chromosomal information obtained from the remaining embryo. We found that the new technique generated information about aneuploidy that was not entirely identical to obtained from the biopsied trophectoderm or the remaining embryo. As the origins of the DNA extracted from the three sample types were not the same, the significance and interpretation of each result would have its own meaning. The possible implications derived from the ECB results as well as those from cell biopsy were discussed. The effectiveness of this new approach in selecting the best embryo for uterine implantation awaits further long term evaluation.