A capture-based method of prenatal cell-free DNA screening for autosomal recessive non-syndromic hearing loss.

OBJECTIVE: This study aimed to develop and validate a prenatal cell-free DNA (cfDNA) screening method that uses capture-based enrichment to genotype fetal autosomal recessive disorders. This method was applied in pregnancies at high risk of autosomal recessive non-syndromic hearing loss (ARNSHL) to assess its accuracy and effectiveness. METHODS: This assay measured the allele counts in both white blood cell DNA and cfDNA from the blood samples of pregnant women using a capture-based next-generation sequencing method. It then applied a binomial model to infer the fetal genotypes with the maximum likelihood. Ninety-four pregnant couples that were carriers of variants of ARNSHL in GJB2 or SLC26A4 were enrolled. The fetal genotypes deduced using this screening method were compared with the results of genetic diagnosis using amniocentesis. RESULTS: Of the 94 couples, 65 carried more than one variant, resulting in 170 single-nucleotide polymorphism (SNP) loci to be inferred in the fetuses. Of the 170 fetal SNP genotypes, 150 (88.2%) had high confidence calls and 139 (92.7%) of these matched the genotypes obtained by amniocentesis result. Out of the remaining 20 (11.8%) cases with low-confidence calls, only 14 (70.0%) were concordant with genetic diagnosis using amniocentesis. The concordance rate was 100% for sites where the maternal genotype was wild-type homozygous. The discordance was site-biased, with each locus showing a consistent direction of discordance. Genetic diagnosis identified a total of 19 wild-type homozygotes, 46 heterozygotes, 19 compound heterozygotes, and 10 pathogenic homozygotes. This screening method correctly genotyped 81.9% (77/94) of fetuses and demonstrated a sensitivity of 89.7% and a specificity of 89.2% for correctly identifying ARNSHL. CONCLUSION: This capture-based method of prenatal screening by cfDNA demonstrated strong potential for fetal genotyping of autosomal recessive disorders.

Novel copy number variations and phenotypes of infantile epileptic spasms syndrome.

We summarize the copy number variations (CNVs) and phenotype spectrum of infantile epileptic spasms syndrome (IESS) in a Chinese cohort. The CNVs were identified by genomic copy number variation sequencing. The CNVs and clinical data were analyzed. 74 IESS children with CNVs were enrolled. 35 kinds of CNVs were identified. There were 11 deletions and 5 duplications not reported previously in IESS, including 2 CNVs not reported in epilepsy. 87.8% were de novo, 9.5% were inherited from mother and 2.7% from father. Mosaicism occurred in one patient with Xq21.31q25 duplication. 16.2% (12/74) were 1p36 deletion, and 20.3% (15/74) were 15q11-q13 duplication. The age of seizure onset ranged from 17 days to 24 months. Seizure types included epileptic spasms, focal seizures, tonic seizures, and myoclonic seizures. All patients displayed developmental delay. Additional features included craniofacial anomaly, microcephaly, congenital heart defects, and hemangioma. 29.7% of patients were seizure-free for more than 12 months, and 70.3% still had seizures after trying 2 or more anti-seizure medications. In conclusion, CNVs is a prominent etiology of IESS. 1p36 deletion and 15q duplication occurred most frequently. CNV detection should be performed in patients with IESS of unknown causes, especially in children with craniofacial anomalies and microcephaly.

A robust microsatellite instability detection model for unpaired colorectal cancer tissue samples.

BACKGROUND: Microsatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples. METHODS: From January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR. RESULTS: We first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes ( MLH1 , MSH2 , MSH6 , and PMS2 ). CONCLUSION: MSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.

Establishment and validation of a multigene model to predict the risk of relapse in hormone receptor-positive early-stage Chinese breast cancer patients.

BACKGROUND: Breast cancer patients who are positive for hormone receptor typically exhibit a favorable prognosis. It is controversial whether chemotherapy is necessary for them after surgery. Our study aimed to establish a multigene model to predict the relapse of hormone receptor-positive early-stage Chinese breast cancer after surgery and direct individualized application of chemotherapy in breast cancer patients after surgery. METHODS: In this study, differentially expressed genes (DEGs) were identified between relapse and nonrelapse breast cancer groups based on RNA sequencing. Gene set enrichment analysis (GSEA) was performed to identify potential relapse-relevant pathways. CIBERSORT and Microenvironment Cell Populations-counter algorithms were used to analyze immune infiltration. The least absolute shrinkage and selection operator (LASSO) regression, log-rank tests, and multiple Cox regression were performed to identify prognostic signatures. A predictive model was developed and validated based on Kaplan-Meier analysis, receiver operating characteristic curve (ROC). RESULTS: A total of 234 out of 487 patients were enrolled in this study, and 1588 DEGs were identified between the relapse and nonrelapse groups. GSEA results showed that immune-related pathways were enriched in the nonrelapse group, whereas cell cycle- and metabolism-relevant pathways were enriched in the relapse group. A predictive model was developed using three genes ( CKMT1B , SMR3B , and OR11M1P ) generated from the LASSO regression. The model stratified breast cancer patients into high- and low-risk subgroups with significantly different prognostic statuses, and our model was independent of other clinical factors. Time-dependent ROC showed high predictive performance of the model. CONCLUSIONS: A multigene model was established from RNA-sequencing data to direct risk classification and predict relapse of hormone receptor-positive breast cancer in Chinese patients. Utilization of the model could provide individualized evaluation of chemotherapy after surgery for breast cancer patients.

A new and improved method of library preparation for non-invasive prenatal testing: plasma to library express technology.

OBJECTIVES: This study aims to develop a novel library preparation method, plasma to library express technology (PLET), to construct next-generation sequencing (NGS) libraries directly from plasma without cell-free DNA (cfDNA) isolation. METHODS: Peripheral blood samples (600) were obtained from a retrospective cohort of 300 pregnant women prior to invasive diagnostic testing. The samples were subsequently distributed between library preparation methodologies, with 300 samples prepared by PLET and 300 by conventional methods for non-invasive prenatal testing (NIPT) to screen for common trisomies using low-pass whole genome next generation sequencing. RESULTS: NIPT conducted on PLET libraries demonstrated comparable metrics to libraries prepared using conventional methods, including 100% sensitivity and specificity. CONCLUSIONS: Our study demonstrates the potential utility of PLET in the clinical setting and highlights its significant advantages, including dramatically reduced process complexity and markedly decreased turnaround time.

RJAfinder: An automated tool for quantification of responding to joint attention behaviors in autism spectrum disorder using eye tracking data.

Deficits in responding to joint attention (RJA) are early symptoms of autism spectrum disorder (ASD). Currently, no automated tools exist for identifying and quantifying RJA behaviors. A few eye tracking studies have investigated RJA in ASD children but have produced conflicting results. In addition, little is known about the trajectory of RJA development through developmental age. Here, a new video was designed including 12 clips of an actor pointing to or looking at an object. Eye tracking technology was used to monitor RJA in three groups: 143 ASD children assessed with the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) (4-7 years old), 113 age- and gender-matched typically developing children (TDC), and 43 typically developing adults (TDA) (19-32 years old). RJAfinder was developed in R and MATLAB to quantify RJA events from the eye tracking data. RJA events were compared among the three groups. Spearman correlation coefficients between total number of RJA events in ASD and the Social Responsiveness Scale (SRS) scores were calculated. A logistic regression model was built using the average valid sampling rate and the total number of RJA events as two predictive variables to classify ASD and TDC groups. ASD children displayed statistically significantly less RJA events than the TDC and TDA groups with medium-to-large-sized effects. ASD and TDC children both displayed more RJA events in response to pointing stimuli than to looking stimuli. Our logistic regression model predicted ASD tendency with 0.76 accuracy in the testing set. RJA ability improved more slowly between the ages of 4-7 years old in the ASD group than in the TDC group. In ASD children, RJA ability showed negative correlation with SRS total T-score as well as the scores of five subdomains. Our study provides an automated tool for quantifying RJA and insights for the study of RJA in ASD children, which may help improve ASD screening, subtyping, and behavior interventions.

Genetic and Clinicopathologic Characteristics of Papillary Thyroid Carcinoma in the Chinese Population: High BRAF Mutation Allele Frequency, Multiple Driver Gene Mutations, and RET Fusion May Indicate More Advanced TN Stage.

PURPOSE: To describe the genetic landscape and clinical characteristics of Chinese patients diagnosed with papillary thyroid cancer (PTC) and to determine which high-risk genetic characteristics suggest a likelihood of lymph node metastasis (LNM) and lateral lymph node metastasis (LLNM). PATIENTS AND METHODS: Data from previously untreated patients with PTC collected between May 2018 and December 2020 from 14 hospitals in China were analyzed retrospectively. High-risk pathologic characteristics were defined as T3/T4, N(+), and N1b(+) stages. All patients were tested for 57 genes by second-generation sequencing. The t-test, chi-square test, and Fisher's exact test were performed for statistical analysis. RESULTS: Overall, 395 patients were enrolled in this study. The prevalence of BRAF mutation was 78.53%. BRAF mutant allele frequency (MAF) >16.93% was associated with a significantly higher risk of LNM, LLNM, and T3 + T4 stage compared with a low-risk group, defined by a MAF <2.54% (odd ratios [ORs] for each risk=3.38, 3.46, and 8.54, respectively), and an intermediate-risk group, defined by a MAF of 2.54% to 16.93% (ORs=2.04, 2.07, and 4.07, respectively). The population with RET fusion had higher T, N, and N1b stages (ORs for each stage=10.40, 7.60, and 8.77, respectively) compared with a RET-negative population. Similar conclusions about T, N, and N1b stages were observed in relation to multiple driver gene mutations (ORs for each stage=7.48, 2.80, and 7.04, respectively) compared with population without multiple driver mutations. These genetic characteristics may be suggestive of high clinical risk. However, regardless of genetic profiles, patients younger than age 45 years had greater rates of LNM and LLNM. CONCLUSION: The main driver gene in this study, BRAF, differs significantly between the United States (79% vs 51%) and other countries. The Chinese population in this study that experienced more aggressive tumor biology had a BRAF MAF greater than 16.93%, exhibited RET fusion events, and had multiple driver gene mutations; thus, these traits may be considered high-risk genetic characteristics in PTC that could warrant aggressive treatment in such population.

Adaptive parameter of standard deviation enhances the power of noninvasive prenatal screens.

PURPOSE: Traditional Z-test methods during noninvasive prenatal screens (NIPS) use the fixed parameter of standard deviation (SD), which ignores the influence of actual sequencing read counts of a sample on the results. The aim of this study is to eliminate the influence of the sequencing depth of individual samples on the results and enhance the power of NIPS. METHODS: In this study, we propose an improved NIPS method, which calculates the SD in the Z-score process adaptively according to the actual read count of the test sample. Our approach obtained the SD linear fitting function along with the read count with a large number of reference samples, in which SD and read count fit well. The effectiveness of our enhanced NIPS method was evaluated on three common trisomy syndromes and five recurrent CNV syndromes with 3219 and 6592 samples based on whole genome sequencing of maternal peripheral blood. RESULTS: A total of 3,219 pregnant samples have been used for validating the proposed method on detecting fetal trisomy syndromes (T13, T18, and T21), in which eight false negative (FN) samples have been corrected as true positive (TP) and eight false positive (FP) samples have been fixed as true negative (TN) with our proposed adaptive-SD method. Another 6592 samples were used to compare the two methods on detecting five recurrent fetal copy number variation (CNV) syndromes, in which the FP samples have decreased from 99 to 39. CONCLUSIONS: Our adaptive-SD NIPS method shows more power on detecting both trisomy syndromes and five recurrent CNVs in the pregnant samples with diverse read counts. Besides, our proposed method contributes to lower FP and FN samples than the traditional Z-test method in NIPS. Our results show that our enhanced NIPS methods are effective in detecting both abnormal fetal trisomy syndromes and recurrent CNV syndromes in pregnant women.

[Consensus on technological standards for non-invasive prenatal screening of pathogenic copy number variations by high-throughput sequencing of maternal plasma cell-free DNA].

Genomic disorders caused by pathogenic copy number variation (pCNV) have proven to underlie a significant proportion of birth defects. With technological advance, improvement of bioinformatics analysis procedure, and accumulation of clinical data, non-invasive prenatal screening of pCNV (NIPS-pCNV) by high-throughput sequencing of maternal plasma cell-free DNA has been put to use in clinical settings. Specialized standards for clinical application of NIPS-pCNV are required. Based on the discussion, 10 pCNV-associated diseases with well-defined conditions and 5 common chromosomal aneuploidy syndromes are recommended as the target of screening in this consensus. Meanwhile, a standardized procedure for NIPS-pCNV is also provided, which may facilitate propagation of this technique in clinical settings.

Clinical spectrum and genetic variations of LMNA-related muscular dystrophies in a large cohort of Chinese patients.

BACKGROUND: LMNA-related muscular dystrophy is caused by mutations in LMNA gene. We aimed to identify genetic variations and clinical features in a large cohort of Chinese patients with LMNA mutations in an attempt to establish genotype-phenotype correlation. METHODS: The clinical presentations of patients with LMNA-related muscular dystrophy were recorded using retrospective and prospective cohort study. LMNA mutation analysis was performed by Sanger sequencing or next-generation sequencing. Mosaicism was detected by personal genome machine amplicon deep sequencing for mosaicism. RESULTS: Eighty-four patients were identified to harbour LMNA mutations. Forty-one of those were diagnosed with LMNA-related congenital muscular dystrophy (L-CMD), 32 with Emery-Dreifuss muscular dystrophy (EDMD) and 11 with limb-girdle muscular dystrophy type 1B (LGMD1B). We identified 21 novel and 29 known LMNA mutations. Two frequent mutations were identified: c.745C>T and c.1357C>T. A correlation between the location of mutation and the clinical phenotype was observed: mutations affecting the head and coil 2A domains mainly occurred in L-CMD, while the coil 2B and Ig-like domains mainly related to EDMD and LGMD1B. We found somatic mosaicism in one parent of four probands. Muscle biopsies revealed 11 of 20 biopsied L-CMD exhibited inflammatory changes, and muscle cell ultrastructure showed abnormal nuclear morphology. CONCLUSIONS: Our detailed clinical and genetic analysis of 84 patients with LMNA-related muscular dystrophy expands clinical spectrum and broadens genetic variations caused by LMNA mutations. We identified 21 novel and 29 known LMNA mutations and found two frequent mutations. A correlation between the location of mutation and the clinical severity was observed. Preliminary data suggested that low-dose corticosteroid treatment may be effective.

MosaicBase: A Knowledgebase of Postzygotic Mosaic Variants in Noncancer Disease-related and Healthy Human Individuals.

Mosaic variants resulting from postzygotic mutations are prevalent in the human genome and play important roles in human diseases. However, except for cancer-related variants, there is no collection of postzygotic mosaic variants in noncancer disease-related and healthy individuals. Here, we present MosaicBase, a comprehensive database that includes 6698 mosaic variants related to 266 noncancer diseases and 27,991 mosaic variants identified in 422 healthy individuals. Genomic and phenotypic information of each variant was manually extracted and curated from 383 publications. MosaicBase supports the query of variants with Online Mendelian Inheritance in Man (OMIM) entries, genomic coordinates, gene symbols, or Entrez IDs. We also provide an integrated genome browser for users to easily access mosaic variants and their related annotations for any genomic region. By analyzing the variants collected in MosaicBase, we find that mosaic variants that directly contribute to disease phenotype show features distinct from those of variants in individuals with mild or no phenotypes, in terms of their genomic distribution, mutation signatures, and fraction of mutant cells. MosaicBase will not only assist clinicians in genetic counseling and diagnosis but also provide a useful resource to understand the genomic baseline of postzygotic mutations in the general human population. MosaicBase is publicly available at http://mosaicbase.com/ or http://49.4.21.8:8000.

Concordance of Genomic Alterations between Circulating Tumor DNA and Matched Tumor Tissue in Chinese Patients with Breast Cancer.

PURPOSE: Circulating tumor DNA (ctDNA) served as a noninvasive method with less side effects using peripheral blood. Given the studies on concordance rate between liquid and solid biopsies in Chinese breast cancer (BC) patients were limited, we sought to examine the concordance rate of different kinds of genomic alterations between paired tissue biopsies and ctDNA samples in Chinese BC cohorts. MATERIALS AND METHODS: In this study, we analyzed the genomic alteration profiles of 81 solid BC samples and 41 liquid BC samples. The concordance across 136 genes was evaluated. RESULTS: The median mutation counts per sample in 41 ctDNA samples was higher than the median in 81 tissue samples (p=0.0254; Wilcoxon rank sum test). For mutation at the protein-coding level, 39.0% (16/41) samples had at least one concordant mutation in two biopsies. 20.0% tissue-derived mutations could be detected via ctDNA-based sequencing, whereas 11.7% ctDNA-derived mutations could be found in paired tissues. At gene amplification level, the overall concordant rate was 68.3% (28/41). The concordant rate at gene level for each patient ranged from 83.8% (114/136) to 99.3% (135/136). And, the mean level of variant allele frequency (VAF) for concordant mutations in ctDNA was statistically higher than that for the discordant ones (p < 0.001; Wilcoxon rank sum test). Across five representative genes, the overall sensitivity and specificity were 49.0% and 85.9%, respectively. CONCLUSION: Our results indicated that ctDNA could provide complementary information on genetic characterizations in detecting single nucleotide variants (SNVs) and insertions and deletions (InDels).

A novel NGS-based microsatellite instability (MSI) status classifier with 9 loci for colorectal cancer patients.

BACKGROUND: With the recent emergence of immune checkpoint inhibitors, microsatellite instability (MSI) status has become an important biomarker for immune checkpoint blockade therapy. There are growing technical demands for the integration of different genomic alterations profiling including MSI analysis in a single assay for full use of the limited tissues. METHODS: Tumor and paired control samples from 64 patients with primary colorectal cancer were enrolled in this study, including 14 MSI-high (MSI-H) cases and 50 microsatellite stable (MSS) cases determined by MSI-PCR. All the samples were sequenced by a customized NGS panel covering 2.2 MB. A training dataset of 28 samples was used for selection of microsatellite loci and a novel NGS-based MSI status classifier, USCI-msi, was developed. NGS-based MSI status, single nucleotide variant (SNV) and tumor mutation burden (TMB) were detected for all patients. Most of the patients were also independently detected by immunohistochemistry (IHC) staining. RESULTS: A 9-loci model for detecting microsatellite instability was able to correctly predict MSI status with 100% sensitivity and specificity compared with MSI-PCR, and 84.3% overall concordance with IHC staining. Mutations in cancer driver genes (APC, TP53, and KRAS) were dispersed in MSI-H and MSS cases, while BRAF p.V600E and frameshifts in TCF7L2 gene occurred only in MSI-H cases. Mismatch repair (MMR)-related genes are highly mutated in MSI-H samples. CONCLUSION: We established a new NGS-based MSI classifier, USCI-msi, with as few as 9 microsatellite loci for detecting MSI status in CRC cases. This approach possesses 100% sensitivity and specificity, and performed robustly in samples with low tumor purity.

Inferring fetal fractions from read heterozygosity empowers the noninvasive prenatal screening.

PURPOSE: Fetal fraction (FF) is the percent of cell-free DNA (cfDNA) in the mother's peripheral blood that is of fetal origin, which plays a pivotal role in noninvasive prenatal screening (NIPS). We present a method that can reliably estimate FFs by examining autosome single-nucleotide polymorphisms (SNPs). METHODS: Even at a very low sequencing depth, there are plenty of SNPs covered by more than one read. At those SNPs, we define read heterozygosity and demonstrate that the percent of read heterozygosity is a function of FF, which allows FF to be inferred. RESULTS: We first demonstrated the effectiveness of our method in inferring FF. Then we used the inferred FF as an informative alternative prior to computing Bayes factors to test for aneuploidy, and observed better power than the Z-test. In analysis of clinical samples, we were able to identify female-male twins thanks to the accurate FF inference. CONCLUSION: Knowing FF improves efficacy of NIPS. It brings a powerful Bayesian method, allows "no call" for samples with small FFs, renders screening for XXY syndrome simpler, and permits an adaptive design to sequence at a higher depth for samples with small FFs.

Correction: Inferring fetal fractions from read heterozygosity empowers the noninvasive prenatal screening.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Somatic LINE-1 retrotransposition in cortical neurons and non-brain tissues of Rett patients and healthy individuals.

Mounting evidence supports that LINE-1 (L1) retrotransposition can occur postzygotically in healthy and diseased human tissues, contributing to genomic mosaicism in the brain and other somatic tissues of an individual. However, the genomic distribution of somatic human-specific LINE-1 (L1Hs) insertions and their potential impact on carrier cells remain unclear. Here, using a PCR-based targeted bulk sequencing approach, we profiled 9,181 somatic insertions from 20 postmortem tissues from five Rett patients and their matched healthy controls. We identified and validated somatic L1Hs insertions in both cortical neurons and non-brain tissues. In Rett patients, somatic insertions were significantly depleted in exons-mainly contributed by long genes-than healthy controls, implying that cells carrying MECP2 mutations might be defenseless against a second exonic L1Hs insertion. We observed a significant increase of somatic L1Hs insertions in the brain compared with non-brain tissues from the same individual. Compared to germline insertions, somatic insertions were less sense-depleted to transcripts, indicating that they underwent weaker selective pressure on the orientation of insertion. Our observations demonstrate that somatic L1Hs insertions contribute to genomic diversity and MeCP2 dysfunction alters their genomic patterns in Rett patients.

ATP1A3 mosaicism in families with alternating hemiplegia of childhood.

Alternating hemiplegia of childhood (AHC) is a rare and severe neurodevelopmental disorder characterized by recurrent hemiplegic episodes. Most AHC cases are sporadic and caused by de novo ATP1A3 pathogenic variants. In this study, the aim was to identify the origin of ATP1A3 pathogenic variants in a Chinese cohort. In 105 probands including 101 sporadic and 4 familial cases, 98 patients with ATP1A3 pathogenic variants were identified, and 96.8% were confirmed as de novo. Micro-droplet digital polymerase chain reaction was applied for detecting ATP1A3 mosaicism in 80 available families. In blood samples, four asymptomatic parents, including two paternal and two maternal, and one proband with a milder phenotype were identified as mosaicism. Six (7.5%) parental mosaicisms were identified in multiple tissues, including four previously identified in blood and two additional cases identified from paternal sperms. Mosaicism was identified in multiple tissues with varied mutant allele fractions (MAFs, 0.03%-33.03%). The results suggested that MAF of mosaicism may be related to phenotype severity. This is the first systematic report of ATP1A3 mosaicism in AHC and showed mosaicism as an unrecognized source of previously considered "de novo" AHC. Identifying ATP1A3 mosaicism provides more evidence for estimating recurrence risk and has implications in genetic counseling of AHC.

Correction: Genomic mosaicism in the pathogenesis and inheritance of a Rett syndrome cohort.

The second author Jiarui Li is now listed as a co-first author according to her contribution to this paper. The list of authors who contributed equally now reads: Qingping Zhang, Xiaoxu Yang, Jiaping Wang, and Jiarui Li. This has now been corrected in both the PDF and HTML versions of the Article.

Targeted resequencing of 358 candidate genes for autism spectrum disorder in a Chinese cohort reveals diagnostic potential and genotype-phenotype correlations.

Autism spectrum disorder (ASD) is a childhood neuropsychiatric disorder with a complex genetic architecture. The diagnostic potential of a targeted panel of ASD genes has only been evaluated in small cohorts to date and is especially understudied in the Chinese population. Here, we designed a capture panel with 358 genes (111 syndromic and 247 nonsyndromic) for ASD and sequenced a Chinese cohort of 539 cases evaluated with the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) as well as 512 controls. ASD cases were found to carry significantly more ultra-rare functional variants than controls. A subset of 78 syndromic and 54 nonsyndromic genes was the most significantly associated and should be given high priority in the future screening of ASD patients. Pathogenic and likely pathogenic variants were detected in 9.5% of cases. Variants in SHANK3 and SHANK2 were the most frequent, especially in females, and occurred in 1.2% of cases. Duplications of 15q11-13 were detected in 0.8% of cases. Variants in CNTNAP2 and MEF2C were correlated with epilepsy/tics in cases. Our findings reveal the diagnostic potential of ASD genetic panel testing and new insights regarding the variant spectrum. Genotype-phenotype correlations may facilitate the diagnosis and management of ASD.

Mosaicism and incomplete penetrance of PCDH19 mutations.

BACKGROUND: Mutations in the PCDH19 gene have mainly been reported in female patients with epilepsy. To date, PCDH19 mutations have been reported in hundreds of females and only in 10 mosaic male epileptic patients with mosaicism. OBJECTIVE: We aimed to investigate the occurrence of mosaic PCDH19 mutations in 42 families comprising at least one patient with PCDH19-related epilepsy. METHODS: Two male patients with mosaic PCDH19 variants were identified using targeted next-generation sequencing. Forty female patients with PCDH19 variants were identified by Sanger sequencing and Multiple Ligation Probe Amplification (MLPA). Microdroplet digital PCR was used to quantify the mutant allelic fractions (MAFs) in 20 families with PCDH19 variants. RESULTS: Five mosaic individuals, four males and one female, were identified in total. Mosaic variant was confirmed in multiple somatic tissues from one male patient and in blood from the other male patient. Among 22 female patients harbouring a newly occurred PCDH19 variant identified by Sanger sequencing and MLPA, Sanger sequencing revealed two mosaic fathers (9%, 2/22), one with two affected daughters and the other with an affected child. Two asymptomatic mosaic fathers were confirmed as gonosomal mosaicism, with MAFs ranging from 4.16% to 37.38% and from 1.27% to 19.13%, respectively. In 11 families with apparent de novo variants, 1 female patient was identified as a mosaic with a blood MAF of 26.72%. CONCLUSION: Our study provides new insights into phenotype-genotype correlations in PCDH19 related epilepsy and the finding of high-frequency mosaicism has important implications for genetic counselling.