Objective: Absence of homozygosity (AOH) is a genetic characteristic known to cause human diseases mainly through autosomal recessive or imprinting mechanisms. The importance and necessity of accurate AOH detection has become more clinically significant in recent years. However, it remains a challenging task for sequencing-based methods thus far. Methods: In this study, we developed and optimized a new bioinformatic algorithm based on the assessment of minimum sequencing coverage, optimal bin size, the Z-score threshold of four types of allele count and the frequency for accurate genotyping using 28 AOH negative samples, and redefined the AOH detection cutoff value. We showed the performance of chromosome analysis by five-fold coverage whole genome sequencing (CMA-seq) for AOH identification in 27 typical prenatal/postnatal AOH positive samples, which were previously confirmed by chromosomal microarray analysis with single nucleotide polymorphism array (CMA/SNP array). Results: The blinded study indicated that for all three forms of AOH, including whole genomic AOH, single chromosomal AOH and segmental AOH, and all kinds of sample types, including chorionic villus sampling, amniotic fluid, cord blood, peripheral blood and abortive tissue, CMA-seq showed equivalent detection power to that of routine CMA/SNP arrays (750K). The subtle difference between the two methods is that CMA-seq is prone to detect small inconsecutive AOHs, while CMA/SNP array reports it as a whole. Conclusion: Based on our newly developed bioinformatic algorithm, it is feasible to detect clinically significant AOH using CMA-seq in prenatal diagnosis.

Objective: Mosaicism is a common biological phenomenon in organisms and has been reported in many types of chromosome abnormalities, including the absence of heterozygosity (AOH). Due to the detection limitations of the sequencing approach, mosaic AOH events are rarely assessed in clinical cases. Herein, we report the performance of mosaic AOH identification using a low-pass (5~8-fold) WGS method (termed ‘CMA-seq’, an abbreviation for ‘Chromosome Analysis by Sequencing’) in fetal genetic diagnosis. Methods: Thirty AOH-negative, eleven constitutional AOH, and three mosaic AOH samples were collected as training data sets to develop the algorithm and evaluate the suitable thresholds for distinguishing mosaic AOH. Twenty-four new chromosomal aberrant cases, along with sixteen constitutional AOH samples, which were previously ascertained via the SNP-array-based method, were used as a validation data set to measure the performance in terms of sensitivity and specificity of this algorithm. Results: A new statistic, ‘D-value’, was implemented to identify and distinguish constitutional and mosaic AOH events. The reporting thresholds for constitutional and mosaic AOH were also established. In the validation set consisting of 24 new cases, seven constitutional AOH cases and 1 mosaic AOH case were successfully identified, indicating that the results were consistent with those of the SNP-array-based method. The results of all sixteen constitutional AOH validation samples also met the threshold requirements. Conclusions: In this study, we developed a new bioinformatic algorithm to accurately distinguish mosaic AOH from constitutional AOH by low-pass WGS. However, due to the small sample size of the training data set, the algorithm proposed in this manuscript still needs further refinements.

The aim of this study was to investigate the detection capability of medium-coverage whole-genome sequencing for chromosomal mosaicism in prenatal diagnosis, and to evaluate the pregnancy outcome of mosaicism. Thirty-four prenatal samples with chromosomal mosaicism diagnosed by chromosomal microarray analysis with single nucleotide polymorphism (CMA/SNP) were included and subsequently subjected to chromosome analysis by medium-coverage whole-genome sequencing (CMA-seq) for back-to-back comparison. The results of karyotyping or fluorescence in situ hybridization (FISH) were reviewed for additional validation. Data on pregnancy outcomes were collected to analyze the effect of mosaicism on fetuses. In total, 13 mosaic autosomal trisomies, 9 sex chromosome aneuploidies (SCAs), and 12 mosaic copy number variants (CNVs) (11 pathogenic or likely pathogenic) were detected by CMA-seq. Twenty-six cases were classified as having high-level (≥ 30%) mosaicism, and 8 were classified as having low-level (< 30%) mosaicism. The results of mosaicism detected by CMA-seq were in 100% agreement with those of CMA/SNP. Among the 25 cases with consistent FISH or karyotyping results, 4 had three cell lines, and 8 had mosaicism involving complex structural aberrations. Except for 2 mosaic SCAs and 1 mosaic CNV of uncertain significance, all pregnancies in the high-level group were terminated, whereas most fetuses without ultrasound abnormalities in the low-level group were delivered. The efficiency of CMA-seq in detecting mosaicism is comparable to that of CMA/SNP. Combining cytogenetic and molecular genetic methods can provide a comprehensive interpretation of mosaicism. The mosaic level, pathogenicity of chromosomal aberrations, and ultrasound findings are the main factors for prenatal decision-making.

Kagami–Ogata syndrome (KOS) and Temple syndrome (TS) are two imprinting disorders characterized by the absence or reduced expression of maternal or paternal genes in the chromosome 14q32 region, respectively. We present a rare prenatally diagnosed case of recurrent KOS inherited from a mother affected by TS. The woman’s two affected pregnancies exhibited recurrent manifestations of prenatal overgrowth, polyhydramnios, and omphalocele, as well as a small bell-shaped thorax with coat-hanger ribs postnatally. Prenatal genetic testing using a single-nucleotide polymorphism array detected a 268.2-kb deletion in the chromosome 14q32 imprinted region inherited from the mother, leading to the diagnosis of KOS. Additionally, the woman carried a de novo deletion in the paternal chromosome 14q32 imprinted region and presented with short stature and small hands and feet, indicating a diagnosis of TS. Given the rarity of KOS as an imprinting disorder, accurate prenatal diagnosis of this rare imprinting disorder depends on two factors: (1) increasing clinician recognition of the clinical phenotype and related genetic mechanism, and (2) emphasizing the importance of imprinted regions in the CMA workflow for laboratory analysis.

The 15q11.2 BP1-BP2 microdeletion (Burnside-Butler syndrome) is a recurrent copy number variation associated with variable neurodevelopmental outcomes. Its incomplete penetrance and broad phenotypic spectrum challenge prenatal genetic counseling. This study aimed to characterize prenatal and postnatal clinical outcomes associated with the 15q11.2 BP1-BP2 microdeletion and to provide evidence to inform prenatal genetic counseling. Among 5246 high-risk pregnancies undergoing invasive prenatal diagnosis at a single center between January 2022 and February 2026, 25 fetuses with 15q11.2 BP1-BP2 microdeletion were identified by chromosomal microarray analysis. Of these, 9 fetuses showed soft ultrasound markers, 3 had fetal growth restriction, and 3 had major structural anomalies. Five pregnancies were electively terminated, whereas 20 were continued. Postnatal follow-up ranging from 3 months to 3 years and 8 months revealed no developmental or growth abnormalities among live-born children (penetrance 0%). A literature review of prenatally diagnosed cases showed that 65 of 127 fetuses had abnormal prenatal ultrasound findings, while postnatal phenotypic abnormalities were reported in 12 of 77 live-born children. When the analysis was restricted to cases with standardized pediatric follow-up (n = 55), the penetrance decreased to 5.5%. Our data indicate that 15q11.2 BP1-BP2 microdeletion generally has favorable early postnatal outcomes. Given the low penetrance observed in prenatally ascertained cohorts and the mild effect size of this copy number variation, our findings suggest that the clinical implications of routinely reporting isolated 15q11.2 BP1-BP2 microdeletion in the prenatal setting may need to be interpreted with caution. Longer-term follow-up studies remain necessary to further clarify the neurodevelopmental impact of this microdeletion.

This study aimed to resolve diagnostic discrepancies in which expanded carrier screening (ECS) in a healthy population suggested hemophilia A carrier status due to F8 intron 22 inversion (Inv22), while further analysis revealed a true distal Xq28 duplication. We evaluated the clinical utility of integrated genetic technologies in distinguishing between these two entities. A healthy pregnant woman underwent ECS, with long-distance polymerase chain reaction (LD-PCR) specifically employed for F8 Inv22 detection. Given the identified heterozygote of F8 inversion, prenatal diagnosis was conducted, integrating LD-PCR, single-nucleotide polymorphism array (SNP array), optical genome mapping (OGM), third-generation long-read sequencing, and X-chromosome inactivation (XCI) analysis to characterize the variant thoroughly. Initial LD-PCR suggested F8 Inv22 in both the fetus and the mother. SNP array revealed a 0.5-Mb Xq28 duplication spanning F8 exons 1–22. OGM and long-read sequencing confirmed the occurrence of duplication while excluding the F8 inversion, revealing int22h-1/int22h-2-mediated nonallelic homologous recombination as the mechanism. XCI analysis revealed skewed inactivation (18.1%) of the duplicated allele in the asymptomatic mother, whereas the fetus exhibited a random XCI pattern. This study highlights a critical diagnostic pitfall: even well-established techniques such as LD-PCR for F8 Inv22 may yield misleading results when applied to general population screening beyond their original diagnostic context. This underscores the importance of accurate interpretation of ECS results through appropriate analytic validation. We propose a tiered strategy: LD-PCR screening followed by OGM or long-read sequencing to differentiate F8 inversions from Xq28 duplications in the general population.