Preimplantation genetic testing for monogenic diseases (PGT-M) can effectively interrupt the transmission of genetic diseases from parents to the offspring before pregnancy. In China, there are over ten million individuals afflicted with monogenic disorders. This literature review summarizes the development of PGT-M in China for the past 24 years, covering the general steps such as the indications and contraindications, genetic and reproductive counselling, biopsy methods, detecting techniques and strategies during PGT-M application in China. The ethical considerations of PGT-M are also be emphasized, including sexual selection, transferring for mosaic embryos, the three-parent baby, and the different opinions for serious adult-onset conditions. Some key policies of the Chinese government for the application of PGT-M are also considered. Methods for regulation of this technique, as well as specific management to increase the accuracy and reliability of PGT-M, are regarded as priority issues in China. The third-generation sequencing and variants testing from RNA level, and non-invasive preimplantation genetic testing using blastocoel fluid and free DNA particles within spent blastocyst medium might be potential techniques and strategies for PGT-M in future.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a rare, autosomal recessive, hereditary neurodegenerative disorder. It is characterized mainly by early onset progressive encephalopathy, concomitant with an increase in interferon-α levels in the cerebrospinal fluid. Preimplantation genetic testing (PGT) is a procedure that could be used to choose unaffected embryos for transfer after analysis of biopsied cells, which prevents at-risk couples from facing the risk of pregnancy termination.
METHODS: Trio-based whole exome sequencing, karyotyping and chromosomal microarray analysis were used to determine the pathogenic mutations for the family. To block the inheritance of the disease, multiple annealing and looping-based amplification cycles was used for whole genome amplification of the biopsied trophectoderm cells. Sanger sequencing and next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping were used to detect the state of the gene mutations. Copy number variation (CNV) analysis was also carried out to prevent embryonic chromosomal abnormalities. Prenatal diagnosis was preformed to verify the PGT outcomes.
RESULTS: A novel compound heterozygous mutation in TREX1 gene was found in the proband causing AGS. A total of 3 blastocysts formed after intracytoplasmic sperm injection were biopsied. After genetic analyses, an embryo harbored a heterozygous mutation in TREX1 and without CNV was transferred. A healthy baby was born at 38th weeks and prenatal diagnosis results confirmed the accuracy of PGT.
CONCLUSIONS: In this study, we identified two novel pathogenic mutations in TREX1, which has not been previously reported. Our study extends the mutation spectrum of TREX1 gene and contributes to the molecular diagnosis as well as genetic counseling for AGS. Our results demonstrated that combining NGS-based SNP haplotyping for PGT-M with invasive prenatal diagnosis is an effective approach to block the transmission of AGS and could be applied to prevent other monogenic diseases.

The incidence of urolithiasis (UL) in children has been increasing. Although the pathogenesis of pediatric UL is controversial and remains unclear, multiple monogenic causes of UL have been identified. We aim to investigate the prevalence of inherited UL causes and explore the genotype-phenotype correlation in a Chinese pediatric group. In this study, we analyzed the DNA of 82 pediatric UL patients using exome sequencing (ES). The data of metabolic evaluation and genomic sequencing were subsequently analyzed together. We detected 54 genetic mutations in 12 of 30 UL-related genes. A total of 15 detected variants were described as pathogenic mutations, and 12 mutations were considered likely pathogenic. Molecular diagnoses were made in 21 patients with pathogenic or likely pathogenic variants. Six novel mutations that were not previously reported were identified in this cohort. Calcium oxalate stones were detected in 88.9% cases (8/9) with hyperoxaluria-related mutations, while 80% of individuals (4/5) with cystinuria-causing defects were diagnosed with cystine stones. Our study highlights the significant genetic abnormalities in pediatric UL and demonstrates the diagnostic power of ES for screening patients with UL.

Mucopolysaccharidosis type I (MPS I) is a severe disease caused by loss-of-function mutation variants in the α-L-iduronidase (Idua) gene. In vivo genome editing represents a promising strategy to correct Idua mutations, and has the potential to permanently restore IDUA function over the lifespan of patients. Here, we used adenine base editing to directly convert A > G (TAG>TGG) in a newborn murine model harboring the Idua-W392X mutation, which recapitulates the human condition and is analogous to the highly prevalent human W402X mutation. We engineered a split-intein dual-adeno-associated virus 9 (AAV9) adenine base editor to circumvent the package size limit of AAV vectors. Intravenous injection of the AAV9-base editor system into MPS IH newborn mice led to sustained enzyme expression sufficient for correction of metabolic disease (GAGs substrate accumulation) and prevention of neurobehavioral deficits. We observed a reversion of the W392X mutation in 22.46 ± 6.74% of hepatocytes, 11.18 ± 5.25% of heart and 0.34 ± 0.12% of brain, along with decreased GAGs storage in peripheral organs (liver, spleen, lung and kidney). Collectively, these data showed the promise of a base editing approach to precisely correct a common genetic cause of MPS I in vivo and could be broadly applicable to the treatment of a wide array of monogenic diseases.

Background: Preimplantation genetic test for monogenic disorders (PGT-M) has been used to select genetic disease-free embryos for implantation during in vitro fertilization (IVF) treatment. However, embryos tested by PGT-M have risks of harboring chromosomal aneuploidy. Hence, a universal method to detect monogenic diseases and genomic imbalances is required. Methods: Here, we report a novel PGT-A/M procedure allowing simultaneous detection of monogenic diseases and genomic imbalances in one experiment. Library was prepared in a special way that multiplex polymerase chain reaction (PCR) was integrated into the process of whole genome amplification. The resulting library was used for one-step low-pass whole genome sequencing (WGS) and high-depth target enrichment sequencing (TES). Results: The TAGs-seq PGT-A/M was first validated with genomic DNA (gDNA) and the multiple displacement amplification (MDA) products of a cell line. Over 90% of sequencing reads covered the whole-genome region with around 0.3-0.4 × depth, while around 5.4%-7.3% of reads covered target genes with >10000 × depth. Then, for clinical validation, 54 embryos from 8 women receiving PGT-M of β-thalassemia were tested by the TAGs-seq PGT-A/M. In each embryo, an average of 20.0 million reads with 0.3 × depth of the whole-genome region was analyzed for genomic imbalance, while an average of 0.9 million reads with 11260.0 × depth of the target gene HBB were analyzed for β-thalassemia. Eventually, 18 embryos were identified with genomic imbalance with 81.1% consistency to karyomapping results. 10 embryos contained β-thalassemia with 100% consistency to conventional PGT-M method. Conclusion: TAGs-seq PGT-A/M simultaneously detected genomic imbalance and monogenic disease in embryos without dramatic increase of sequencing data output.

In the past decade, digital PCR (dPCR), as a new nucleic acid absolute quantification technology, has been widely used in clinical research. dPCR does not rely on the standard curve and has a higher tolerance to inhibitors. Therefore, it is more accurate than quantitative real-time PCR (qPCR) for the absolute quantification of target sequences. In this article, we aim to review the application of dPCR in noninvasive prenatal testing (NIPT). We focused on the progress of dPCR in screening and identifying fetal chromosome aneuploidies and monogenic mutations. We introduced some common strategies for dPCR in NIPT and analyzed the advantages and disadvantages of different methods. In addition, we compared dPCR with qPCR and next-generation sequencing, respectively, and described their superiority and shortcomings in clinical applications. Finally, we envisaged what the future of dPCR might be in NIPT. Although dPCR can provide reproducible results with improved accuracy due to the digital detection system, it is essential to combine the merits of dPCR and other molecular techniques to achieve more effective and accurate prenatal diagnostic strategies.

Background: Non-invasive prenatal diagnosis (NIPD) can identify monogenic diseases early during pregnancy with negligible risk to fetus or mother, but the haplotyping methods involved sometimes cannot infer parental inheritance at heterozygous maternal or paternal loci or at loci for which haplotype or genome phasing data are missing. This study was performed to establish a method that can effectively recover the whole fetal genome using maternal plasma cell-free DNA (cfDNA) and parental genomic DNA sequencing data, and validate the method\'s effectiveness in noninvasively detecting single nucleotide variations (SNVs), insertions and deletions (indels). Methods: A Bayesian model was developed to determine fetal genotypes using the plasma cfDNA and parental genomic DNA from five couples of healthy pregnancy. The Bayesian model was further integrated with a haplotype-based method to improve the inference accuracy of fetal genome and prediction outcomes of fetal genotypes. Five pregnancies with high risks of monogenic diseases were used to validate the effectiveness of this haplotype-assisted Bayesian approach for noninvasively detecting indels and pathogenic SNVs in fetus. Results: Analysis of healthy fetuses led to the following accuracies of prediction: maternal homozygous and paternal heterozygous loci, 96.2 ± 5.8%; maternal heterozygous and paternal homozygous loci, 96.2 ± 1.4%; and maternal heterozygous and paternal heterozygous loci, 87.2 ± 4.7%. The respective accuracies of predicting insertions and deletions at these types of loci were 94.6 ± 1.9%, 80.2 ± 4.3%, and 79.3 ± 3.3%. This approach detected pathogenic single nucleotide variations and deletions with an accuracy of 87.5% in five fetuses with monogenic diseases. Conclusions: This approach was more accurate than methods based only on Bayesian inference. Our method may pave the way to accurate and reliable NIPD.

Spina bifida (SB) is the second most common nonlethal congenital malformation. The existence of monogenic SB mouse models and human monogenic syndromes with SB features indicate that human SB may be caused by monogenic genes. We hypothesized that whole exome sequencing (WES) allows identification of potential candidate genes by (i) generating a list of 136 candidate genes for SB, and (ii) by unbiased exome-wide analysis. We generated a list of 136 potential candidate genes from three categories and evaluated WES data of 50 unrelated SB cases for likely deleterious variants in 136 potential candidate genes, and for potential SB candidate genes exome-wide. We identified 6 likely deleterious variants in 6 of the 136 potential SB candidate genes in 6 of the 50 SB cases, whereof 4 genes were derived from mouse models, 1 gene was derived from human nonsyndromic SB, and 1 gene was derived from candidate genes known to cause human syndromic SB. In addition, by unbiased exome-wide analysis, we identified 12 genes as potential candidates for SB. Identification of these 18 potential candidate genes in larger SB cohorts will help decide which ones can be considered as novel monogenic causes of human SB.

OBJECTIVE: Is there a simple and effective method for male patients with genetic disorders in families with no identified haplotype and with Robertsonian translocations to avoid the transfer of embryos carrying translocated chromosomes?
METHODS: Single spermatozoa were separated to identify by next-generation sequencing (NGS) those that were genetically abnormal, to establish a sperm-based single-nucleotide polymorphism (SNP) haplotype. Blastocysts that developed to day 5 or 6 were then biopsied for whole genome amplification and screening for chromosomal aneuploidy. Normal embryos were selected by comparison with a single-sperm-based SNP haplotype and were transferred. The results were verified by second trimester amniocentesis.
RESULTS: Two blastocysts obtained from patients with neurofibroma type 1 (NF1) were found to be normal after NGS according to single-sperm-based SNP haplotype analysis (13 SNP sites). Three and one blastocysts, respectively, were obtained from the patients with Robertsonian translocation. Blastocysts B9 and B7 were found to be normal after NGS according to the single-sperm-based SNP haplotype analysis (12 and 13 SNP sites selected on chromosomes 14 and 22 for the first patient; 12 and 9 SNP sites selected on chromosomes 13 and 14 for the second patient). Successful pregnancies after blastocyst transfer occurred in all three patients. The identification of embryos was verified by mid-trimester amniocentesis. All three patient couples successfully delivered healthy babies.
CONCLUSIONS: This study preliminarily summarized the process of single-sperm-based SNP haplotyping, which could be applied as preimplantation genetic testing for male patients without identified disease-causing haplotypes and with Robertsonian translocations.

Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband\'s samples.
The study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis.
Seventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call.
Targeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.