Validation and depth evaluation of low-pass genome sequencing in prenatal diagnosis using 387 amniotic fluid samples.

BACKGROUND: Low-pass genome sequencing (LP GS) is an alternative to chromosomal microarray analysis (CMA). However, validations of LP GS as a prenatal diagnostic test for amniotic fluid are rare. Moreover, sequencing depth of LP GS in prenatal diagnosis has not been evaluated. OBJECTIVE: The diagnostic performance of LP GS was compared with CMA using 375 amniotic fluid samples. Then, sequencing depth was evaluated by downsampling. RESULTS: CMA and LP GS had the same diagnostic yield (8.3%, 31/375). LP GS showed all copy number variations (CNVs) detected by CMA and six additional variant of uncertain significance CNVs (>100 kb) in samples with negative CMA results; CNV size influenced LP GS detection sensitivity. CNV detection was greatly influenced by sequencing depth when the CNV size was small or the CNV was located in the azoospermia factor c (AZFc) region of the Y chromosome. Large CNVs were less affected by sequencing depth and more stably detected. There were 155 CNVs detected by LP GS with at least a 50% reciprocal overlap with CNVs detected by CMA. With 25 M uniquely aligned high-quality reads (UAHRs), the detection sensitivity for the 155 CNVs was 99.14%. LP GS using samples with 25 M UAHRs showed the same performance as LP GS using total UAHRs. Considering the detection sensitivity, cost and interpretation workload, 25 M UAHRs are optimal for detecting most aneuploidies and microdeletions/microduplications. CONCLUSION: LP GS is a promising, robust alternative to CMA in clinical settings. A total of 25 M UAHRs are sufficient for detecting aneuploidies and most microdeletions/microduplications.

Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

Prenatal diagnosis of Walker-Warburg syndrome due to compound mutations in the B3GALNT2 gene.

BACKGROUND: Congenital hydrocephalus is one of the symptoms of Walker-Warburg syndrome that is attributed to the disruptions of the genes, among which the B3GALNT2 gene is rarely reported. A diagnosis of the Walker-Warburg syndrome depends on the clinical manifestations and the whole-exome sequencing after birth, which is unfavorable for an early diagnosis. METHODS: Walker-Warburg Syndrome was suspected in two families with severe fetal congenital hydrocephalus. Whole-exome sequencing and Sanger sequencing were performed on the affected fetuses. RESULTS: The compound heterozygous variants c.1A>G p.(Met1Val) and c.1151+1G>A, and c.1068dupT p.(D357*) and c.1052 T>A p.(L351*) in the B3GALNT2 gene were identified, which were predicted to be pathogenic and likely pathogenic, respectively. Walker-Warburg syndrome was prenatally diagnosed on the basis of fetal imaging and whole-exome sequencing. CONCLUSIONS: Our findings expand the spectrum of pathogenic mutations in Walker-Warburg syndrome and provide new insights into the prenatal diagnosis of the disease.

Novel deep intronic and frameshift mutations causing a TRIP11-related disorder.

Mutations of the thyroid hormone receptor interactor 11 gene (TRIP11, OMIM: 604505) at 14q32.12 have been associated with the autosomal recessive achondrogenesis type IA (ACG1A, OMIM: 200600) or osteochondrodysplasia (ODCD, OMIM: 184260). In this clinical report of a Chinese family, the mother had two consecutive pregnancies with similar aberrant phenotypes in the fetuses showing severe limb shortening. Whole exome sequencing (WES) of DNA from the second fetus identified a heterozygous frameshift mutation (NM_004239: c.3852delT) of TRIP11. Although this was consistent with the fetal clinical phenotypes, initial review of the WES results implied another novel mutation. To test this, we used high-precision clinical exome sequencing (HPCES) and found a mutation in Intron 18 of TRIP11 (c.5457+77T>G). Moreover, the sequencing depth of this mutation was only 3× that of WES compared with 161× that by HPCES. To ascertain the pathogenesis of the mutation (c.5457+77T>G), RT-PCR conducted using the parents' blood samples showed a 77-bp intronic sequence in the transcripts, which might have encoded for a shortened protein because of early termination due to code shifting. Our study furthers current understanding of deep intron function and provides a novel diagnostic method of deep intragenic mutations in families having two or more consecutive pregnancies with similar aberrant fetal phenotypes.

Efficiency of non-invasive prenatal screening in pregnant women at advanced maternal age.

BACKGROUND: Non-invasive prenatal screening (NIPS) is widely used as the alternative choice for pregnant women at high-risk of fetal aneuploidy. However, whether NIPS has a good detective efficiency for pregnant women at advanced maternal age (AMA) has not been fully studied especially in Chinese women. METHODS: Twenty-nine thousand three hundred forty-three pregnant women at AMA with singleton pregnancy who received NIPS and followed-up were recruited. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), receiver operating characteristic (ROC) curves and the Youden Index for detecting fetal chromosomal aneuploidies were analyzed. The relationship between maternal age and common fetal chromosomal aneuploidy was observed. RESULTS: The sensitivity, specificity, PPV, NPV of NIPS for detecting fetal trisomy 21 were 99.11, 99.96, 90.98, and 100%, respectively. These same parameters for detecting fetal trisomy 18 were 100, 99.94, 67.92, and 100%, respectively. Finally, these parameters for detecting trisomy 13 were 100, 99.96, 27.78, and 100%, respectively. The prevalence of fetal trisomy 21 increased exponentially with maternal age. The high-risk percentage incidence rate of fetal trisomy 21 was significantly higher in the pregnant women at 37 years old or above than that in pregnant women at 35 to 37 years old. (Youden index = 37). CONCLUSION: It is indicated that NIPS is an effective prenatal screening method for pregnant women at AMA.

[Genetic Study and Prenatal Diagnosis of a Family with Thrombocytopenia-Absent Radius (TAR) Syndrome].

OBJECTIVE: To analyze the potential genetic cause of thrombocytopenia-absent radius (TAR) syndrome in a family and provide prenatal diagnosis for them. METHODS: Genetic mutation analysis of the sporadic family with TAR syndrome was performed with chromosome microarray analysis (CMA), quantitative polymerase chain reaction (qPCR) and Sanger sequencing. DNA samples were collected from 4 members of the family, including the proband, her parents and her sister. CMA, qPCR and Sanger sequencing were performed to determine the pathogenic mutation and prenatal diagnosis of the fetus was made accordingly. RESULTS: The proband had a 378 kb genomic heterozygous deletion in 1q21.1, which contained RBM8 A and other genes. c.-21G>A mutation was also found in the RBM8 A of the proband. The above-mentioned microdeletion and mutation were inherited from the mother and father, respectively. Prenatal CMA suggested that the fetus carried a 378 kb microdeletion in 1q21.1, and DNA testing did not find c.-21G>A mutation. CONCLUSION: The heterozygous deletion in 1q21.1 and RBM8 A: c.-21G>A is considered to be the genetic etiology of TAR syndrome in the family. The study provides information for subsequent family genetic counseling and prenatal diagnosis.

Basonuclin 1 deficiency is a cause of primary ovarian insufficiency.

Primary ovarian insufficiency (POI) leads to infertility and premature menopause in young women. The genetic etiology of this disorder remains unknown in most patients. Using whole exome sequencing of a large Chinese POI pedigree, we identified a heterozygous 5 bp deletion inducing a frameshift in BNC1, which is predicted to result in a non-sense-mediated decay or a truncated BNC1 protein. Sanger sequencing identified another BNC1 missense mutation in 4 of 82 idiopathic patients with POI, and the mutation was absent in 332 healthy controls. Transfection of recombinant plasmids with the frameshift mutant and separately with the missense mutant in HEK293T cells led to abnormal nuclear localization. Knockdown of BNC1 was found to reduce BMP15 and p-AKT levels and to inhibit meiosis in oocytes. A female mouse model of the human Bnc1 frameshift mutation exhibited infertility, significantly increased serum follicle-stimulating hormone, decreased ovary size and reduced follicle numbers, consistent with POI. We report haploinsufficiency of BNC1 as an etiology of human autosomal dominant POI.

Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform.

BACKGROUND: Recent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S. METHODS: The artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH). RESULTS: Our SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable. CONCLUSIONS: Our studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.

Is Noninvasive Prenatal Screening Appropriate for Pregnant Women Age 35 or Older In Cases if Isolated Fetal Nasal Bone Abnormalities in The Chinese Han Population?

BACKGROUND Ethnic background may affect the prevalence of nasal bone absence and the length of the nasal bone. This study aimed to elucidate the significance of absent or hypoplastic fetal nasal bone in the Chinese Han population and to formulate an optimal management plan for patients age 35 or older in cases of isolated abnormal fetal nasal bone. MATERIAL AND METHODS We prospectively assigned pregnant women whose fetuses had nasal bone absence or hypoplasia to separate groups according to their choice for noninvasive prenatal screening (NIPS) between January 1, 2013, and December 31, 2018. Demographic data, ultrasound findings, results of conventional maternal serum screening and NIPS, fetal karyotype, pregnancy outcomes, and expenses associated with prenatal testing were recorded. The incidence and odds ratio of nasal bone abnormality and the sensitivity and specificity of different prenatal genetic screening tests were calculated. RESULTS A total of 1946 cases with fetal nasal bone absence or hypoplasia were included. Cases of isolated nasal bone abnormality (1736 cases) were divided into the NIPS group (Gr 1, n=429) and the non-NIPS group (Gr 2, n=1307). Sixty-four cases involved chromosomal abnormality. The sensitivity, specificity, and positive and negative predictive values of NIPS in Gr 1 were 100%, 100%, 100%, and 100%, respectively. The odds ratio of fetal chromosomal abnormalities for isolated fetal nasal bone abnormalities when maternal age was ≥35 was 4.615 (95% CI: 1.592-13.381). The cost-effectiveness ratio of contingent screening (NIPS first) was significantly lower than amniocentesis directly. CONCLUSIONS The nasal bone provides an important marker for chromosome abnormalities in some populations, but to a lesser extent in the Chinese Han population. NIPS is an excellent first option for follow-up among pregnant women age ≥35 in cases of absent or hypoplastic fetal nasal bone in the first trimester ultrasound scan.

[Genetic analysis of a child with Sotos syndrome].

OBJECTIVE: To explore the genetic basis for a child with mentally retardation. METHODS: G-banding karyotyping, single nucleotide polymorphism array (SNP-array) and fluorescence in situ hybridization (FISH) were performed for the child. Karyotyping and FISH were also carried out for her parents. RESULTS: SNP-array has detected a 5077 kb microdeletion at 5q35.2q35.3 and a 4964 kb microduplication at 7q36.2q36.3 in the child. The results were confirmed by FISH. Based on above results, the father was subsequently found to carry a cryptic t(5;7) (q35.2; q36.2) translocation. The child was verified to have inherited a der(5) t(5;7)(q35.2; q36.2) from her father. CONCLUSION: The 5077 kb microdeletion at 5q35.2q35.3 may have predisposed to the Sotos syndrome in the child. SNP-array combined with G-banding karyotyping and FISH can help to detect cryptic chromosomal translocations among patients.

[Genetic diagnosis of a fetus with Dandy-Walker syndrome].

OBJECTIVE: To explore the genetic basis for a fetus with Dandy-Walker malformation. METHODS: G-banding chromosomal karotyping, single nucleotide polymorphism microarray (SNP array) and fluorescence in situ hybridization (FISH) were carried out for the fetus. Chromosomal karyotyping and FISH assay were also carried out for both parents. RESULTS: SNP array has detected a 4266 kb microdeletion at 6p25.3p25.1 in the fetus, which was confirmed by FISH. FISH analysis of the parents demonstrated that the father has carried a cryptic t(6;14) (p25.1;p13) translocation, while the fetus has a der(6)t(6;14)(p25.1;p13) derived the paternal translocation. CONCLUSION: The der(6)t(6;14)(p25.1;p13) probably underlies the Dandy-Walker malformation in the fetus. The 6p25.3p25.1 microdeletion is due to unbalanced gametes produced by the father's cryptic balanced translocation.

[Genetic analysis and clinical phenotype of a family with lymphedema-distichiasis syndrome].

OBJECTIVE: To identify the genetic causes of a family with lymphedema-distichiasis syndrome (LDS). METHODS: The whole exome sequencing was performed in a aborted fetus as the proband, and a candidate gene was identified. Peripheral blood of 8 family members were collected. Genotypic-phenotypic analysis were carried out through PCR amplification and Sanger sequencing. RESULTS: The proband, and the mother, grandmother, uncle, granduncle of the proband all had distichiasis or varix of lower limb carried a FOXC2:c.595dupC frame shift mutation, and other subjects without any significant phenotypes did not present the mutation. CONCLUSIONS: The FOXC2:c.595dupC frame shift mutation is the genetic cause of this family, which can lead to autosomal dominantly LDS, presenting nuchal translucency thickening and hydrops fetal during pregnancy, and the prognosis is usually good.

[Genetic analysis of a mosaic case with low proportion mutation of TSC2 gene].

OBJECTIVE: To perform gene mutation analysis in a patient with atypical clinical manifestations of tuberous sclerosis (TSC) for definite diagnosis. METHODS: Peripheral blood DNA was obtained from a patient with clinically suspected TSC and her parents, and all exons and their flanking sequences of TSC1 and TSC2 genes in the proband were sequenced by whole exome sequencing to determine the candidate pathogenic mutations. At the same time, Sanger sequencing was performed to verify the peripheral blood DNA of the patient and her parents. And the mosaic percentage of the mutation in the proband's somatic cells was detected by the droplet digital PCR method. RESULTS: A heterozygous nonsense mutation c.1096G>T (p.E366*) was identified in the exon 11 of the TSC2 gene, which only had a small mutation peak. A lower percentage of the mutation was found in the DNA of the patient than that in the public database, therefore the possibility of mosaicism might not be excluded. In addition, the droplet digital PCR method demonstrated that the proband was a c.1096G>T mutant mosaicism, and the mosaic percentage was 14%. CONCLUSIONS: The somatic mosaic mutation c.1096G>T (p.e366*) may be responsible for the phenotype of TSC in this patient. And the drop digital PCR is expected to be a diagnostic method for somatic cells mosaicism.

[Prenatal diagnosis of a fetus with Phelan-McDermid syndrome].

OBJECTIVE: To diagnose a fetus with Phelan-McDermid syndrome (PMS) using various techniques. METHODS: Single nucleotide polymorphism array (SNP Array), multiplex ligation-dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH) were applied in conjunction for the prenatal diagnosis of the fetus. RESULTS: SNP Array detected a 4.03 Mb microdeletion at 22q13.31q13.33 in the fetus, which was confirmed by FISH and MLPA. FISH analysis of the parents suggested that the 22q13.31q13.33 deletion has a de novo origin. CONCLUSION: Combined use of various techniques can enable accurate prenatal diagnosis and genetic counseling.

[Genetic analysis of a family of Van der Woude syndrome].

OBJECTIVE: To analyze clinical and genetic features of a family affected with Van der Woude syndrome. METHODS: The umbilical cord blood of the proband and the peripheral blood of the parents were used for the whole exon sequencing to find the candidate gene.Peripheral blood of 9 members of the family were collected for Sanger sequencing verification, bioinformatics analysis and genotype-phenotype correlation analysis. RESULTS: The proband was diagnosed with cleft lip and palate by ultrasound. His father and grandmother had hollow lower lip and all other family members did not have the similar phenotype. A missense c.263A>G (p.N88S) mutation was found in exon 4 of IRF6 gene in the proband, his father and his grandmother.The mutation was not found in other family members. CONCLUSIONS: A missense c.263A>G (p.N88S) mutation in IRF6 gene probably underlies the pathogenesis of Van der Woude syndrome in the family and the mutation has been firstly discovered in China.

[Noninvasive prenatal screening for twin pregnancy: an analysis of 2057 cases].

OBJECTIVE: To analyze the results of noninvasive prenatal screening (NIPS) for fetal chromosome aneuploidy in twin pregnancy. METHODS: A total of 2057 women with twin-pregnancy between 12-26+6 weeks were recruited from Women's Hospital, Zhejiang University School of Medicine, Hangzhou Municipal Women's Hospital and Jiaxing Maternal and Child Health Hospital during February 2015 to August 2018. The cell-free DNA was extracted from the peripheral blood sample for DNA library, and non-invasive prenatal testing (NIPT) was performed by high-throughput sequencing technique. The fetal karyotype analysis or neonatal karyotype analysis was performed in pregnant women with fetal chromosome aneuploidy, and all subjects were followed up. The efficiency of NIPS testing for twin aneuploidy was calculated. RESULTS: NIPS revealed chromosome abnormalities in 11 out of 2057 twin pregnant women, 9 cases were confirmed chromosome abnormalities, 2 cases were normal and no false negative cases. In this screening, the detection rate, sensitivity, specificity, positive predictive value, false positive rate of NIPS were 100.00%, 100.00%, 99.90%, 81.82%, 0.10%. Those were 100.00%, 100.00%, 99.95%, 87.50% and 0.05% for trisomy 21, 100.00%, 100.00%, 100.00%, 100.00%, 0.00% for trisomy18, and the specificity and false positive rate for trisomy13 were 99.95% and 0.05%, respectively. CONCLUSIONS: NIPS can detect fetal chromosomal aneuploidy rapidly and accurately in twin pregnancies,and it is of value in clinical application.

[Single nucleotide polymorphism microarray in prenatal diagnosis of fetuses with absent nasal bone].

OBJECTIVE: To assess the clinical application of single nucleotide polymorphism microarray (SNP array) in prenatal genetic diagnosis for fetuses with absent nasal bone. METHODS: Seventy four fetuses with absent nasal bone detected by prenatal ultrasound scanning were recruited from Women's Hospital, Zhejiang University School of Medicine during June 2015 and October 2018. The chromosome karyotypes analysis and SNP array were performed. The correlation between absent fetal nasal bone and chromosome copy number variants was analyzed. RESULTS: Among 74 fetuses, 19 were detected to have chromosomal abnormalities, including 16 cases of trisomy-21, 1 case of trisomy-18 and two cases of micro-deletion/duplication. Among 46 cases with isolated absence of nasal bone, 3 had trisomy-21, and 1 had a micro-duplication. Absence of nasal bone in association with nuchal translucency thickening had a higher rate of abnormal karyotypes compared with isolated absence of nasal bone (χ2=32.27,P<0.01). CONCLUSIONS: Fetuses with absent nasal bone and nuchal translucency thickening are likely to have chromosome abnormalities, and SNP array testing is recommended to exclude the chromosome abnormalities.

[Application of single nucleotide polymorphism microarray in clinical diagnosis of intellectual disability or retardation].

OBJECTIVE: To assess the clinical application of single nucleotide polymorphism microarray (SNP array) in patients with intellectual disability/developmental delay(ID/DD). METHODS: SNP array was performed to detect genome-wide DNA copy number variants (CNVs) for 145 patients with ID/DD in Women's Hospital, Zhejiang University School of Medicine from January 2013 to June 2018. The CNVs were analyzed by CHAS software and related databases. RESULTS: Among 145 patients, pathogenic chromosomal abnormalities were detected in 32 cases, including 26 cases of pathogenic CNVs and 6 cases of likely pathogenic CNVs. Meanwhile, 18 cases of uncertain clinical significance and 14 cases of likely benign were identified, no significant abnormalities were found in 81 cases (including benign). CONCLUSIONS: SNP array is effective for detecting chromosomal abnormalities in patients with ID/DD with high efficiency and resolution.

[Genetic analysis and prenatal diagnosis of a sporadic family with neurofibromatosis type 1].

OBJECTIVE: To identify pathogenic mutation for a family with neurofibromatosis type 1(NF1) and provide prenatal diagnosis for them. METHODS: Mutation analysis of the sporadic family with NF1 was performed with target captured next generation sequencing and Sanger sequencing. RNA samples were extracted from the lymphocytes of NF1 patient and her parents. RT-PCR and Sanger sequencing were performed to analyze the relative mRNA expression in the samples. Prenatal diagnosis of the pathogenic mutation was offered to the fetus. RESULTS: A novel splicing mutation c.1260+4A>T in the NF1 gene was found in the proband of the family, but was not found in her parents.cDNA sequencing showed that 13 bases inserted into the 3' end of exon 11 in the NF1 gene lead to a frameshift mutation. Prenatal diagnosis suggested that the fetus did not carried the mutant. CONCLUSIONS: The NF1: c.1260+4A>T mutation found in the NF1 patient is considered to be pathogenic, which provides information for family genetic counseling and prenatal diagnosis.

[Genetic analysis of a fetus with multiple malformations caused by complex translocations of four chromosomes].

OBJECTIVE: To conduct genetic analysis in a fetus with complex translocation of four chromosomes. METHODS: G-banded chromosome karyotype analysis, single nucleotide polymorphism array (SNP array) and fluorescence in situ hybridization (FISH) were performed in a fetus with multiple malformations. Peripheral blood chromosome karyotype and FISH were also carried out for the parents. RESULTS: The fetal amniotic fluid karyotype was 46, XY, t(12; 13)(q22; q32). SNP array analysis showed that there were 20 192 kb duplication at 1q42.13q44 and 13 293 kb deletion at 15q26.1q26.3 in the fetus. The results of karyotype and SNP array were inconsistent. FISH analyses on the parental peripheral blood samples demonstrated that the mother was a cryptic 46, XX, t(1; 15)(q42.1; q26.1) translocation. The fetus had inherited 46, XY, t(12; 13)(q22; q32) from his father and der(15)t(1; 15)(q42.1; q26.1) from his mother. CONCLUSIONS: The 1q42.13q44 duplication and 15q26.1q26.3 deletion may have contributed to the abnormal sonographic features of the fetus. The combination of cytogenetic, SNP array and FISH techniques was beneficial for providing an accurate genetic counseling.