Yu Qi

Background: The advent of next‐generation sequencing (NGS) has enhanced the diagnostic efficacy for monogenic diseases, while presenting challenges in achieving consistent diagnoses.Method: We retrospectively analyzed the concordance rate and reasons for the inconsistency between the original diagnostic result from the genetic testing laboratory and the variant validation result from the prenatal diagnostic center. The validation procedure comprised three stages: validation of variant detection, reevaluation of variant classification, and assessment of recurrence risk, which involved verifying the mode of inheritance and parental carriage.Result: In total, 17 (6%) of the 286 families affected by rare monogenic diseases showed different results during the variant validation procedure. These cases comprised four (23.5%) with variant detection errors, 12 (70.5%) with inconsistent interpretation, and one (6%) with non‐Mendelian inheritance patterns. False‐positive NGS results confirmed by Sanger sequencing were related to pseudogenes and GC‐rich regions. The classification of the 17 variants was altered in the 12 cases owing to various factors. The case with an atypical inheritance pattern was originally considered autosomal recessive inheritance, but was diagnosed as maternal uniparental disomy after additional genetic analysis.Conclusion: We underscored the significance of variant validation by prenatal diagnostic centers. Families affected by monogenic diseases with reproductive plans should be referred to prenatal genetic centers as early as possible to avoid different results that may postpone subsequent prenatal diagnosis.

In the postgenomic era, variant interpretation is crucial for diagnosing monogenic diseases, which is the premise of precision medicine. The bottleneck and difficulty of genetic disease diagnosis have switched from the inaccessibility of detection technology to the interpretation of sequencing results. Multiple studies have suggested that the inconsistency rate of interlaboratory variant interpretation is approximately 10~40%. However, many clinicians have not paid enough attention to this area at present. In this review, we summarized the reasons for inconsistency, including classification methodology, information obtained by the interpreter, evidence application, and expert judgement. For clinicians, genetic counsellors, and molecular pathologists, it is necessary to reevaluate genetic reports, especially those supported by old literature and databases in clinical practice. For unresolvable cases, pedigree analysis, collaboration with research labs for functional experiments, and long-term follow-up to combine advanced clinical presentations with updated data and literature are needed.

Background: Variants in the ATP binding cassette protein subfamily D member 1 (ABCD1) gene are known to cause X‐linked adrenoleukodystrophy (X‐ALD). This study focused on the characteristics of ABCD1 variants in Chinese X‐ALD families and elucidated the value of genetic approaches for X‐ALD.Methods: 68 male probands diagnosed as X‐ALD were screened for ABCD1 variants by the Sanger sequencing of polymerase chain reaction (PCR) products and multiplex ligation‐dependent probe amplification (MLPA) combined with long‐range PCR. Prenatal diagnosis was performed in 20 foetuses of 17 probands’ mothers. Descriptive statistics were used to summarise the gene variants and prenatal diagnosis characteristics and outcomes.Results: This study allowed the identification of 61 variants occurring in 68 families, including 58 single nucleotide variants or small deletion/insertion variants and 3 large deletions. Three probands with no variants detected by next‐generation sequencing were found to have variants by PCR‐sequencing. Prenatal diagnosis found that 10 of the 20 foetuses had no variants in ABCD1.Conclusion: PCR primers that do not amplify the pseudogenes must be used for PCR‐sequencing. MLPA combined with long‐range PCR can detect large deletions and insertions, which are usually undetectable by PCR‐sequencing. Prenatal diagnosis could help to prevent the birth of infants with X‐ALD.

The prevalence of CGG repeat expansion mutation in FMR1 gene varies among different populations. In this study, we investigated the prevalence of this mutation in women of reproductive age from northern China. A total of 11,891 pre-conceptional or pregnant women, including 5037 pregnant women and 7357 women with the history of spontaneous abortion or induced abortion due to delayed growth of the embryos, were recruited. The number of CGG repeats in FMR1 was measured by the TRP-PCR method. We also offered genetic counseling and prenatal diagnosis to the women carrying pre-mutation or full mutation alleles. The prevalence of pre-mutation in reproductive women in northern China was 1/410, higher than that in southern China and Korea but lower than that in western countries. We also found that the prevalence of pre-mutation was relatively high (1/320) in women with abortion history. Screening for CGG repeat expansion mutation in FMR1 should be recommended to the women with the history of spontaneous abortion or induced abortion due to delayed growth of the embryos.

Ion. Methods: Two healthy cohorts of 200 Chinese minors (0.1–18.0 years) and 200 adults (18.0–88.0 years) were recruited. Then, they were further categorized into eight age groups. The absolute mtDNA copy number per cell was measured by a quantitative real-time polymerase chain reaction. We subsequently used this range to evaluate mtDNA content in four patients (0.5–4.0 years) with molecularly proven mitochondrial depletion syndromes (MDSs) and 83 cases of mitochondrial disease patients harboring the m.3243A>G mutation. Results: The reference range of mtDNA copy number in peripheral blood was 175–602 copies/cell (mean: 325 copies/cell) in minors and 164–500 copies/cell (mean: 287 copies/cell) in adults. There was a decreasing trend in mtDNA copy number in blood with increasing age, especially in 0–2-year-old and >50-year-old donors. The mean mtDNA copy number level among the mitochondrial disease patients with m.3243A>G mutation was significantly higher than that of healthy controls. The mtDNA content of POLG, DGUOK, TK2, and SUCLA2 genes in blood samples from MDS patients was reduced to 25%, 38%, 32%, and 24%, respectively. Conclusions: We primarily establish the reference intervals of mtDNA copy number, which might contribute to the clinical diagnosis and monitoring of mitochondrial disease....

Large deletions in mitochondrial DNA (mtDNA) may be involved in the pathogenesis of mitochondrial disease. In this study, we investigated the relationship between a 4,977-bp deletion in the mitochondrial genome (ΔmtDNA⁴⁹⁷⁷) and the severity of clinical symptoms in patients with mitochondrial disease lacking known point mutations. A total of 160 patients with mitochondrial disease and 101 healthy controls were recruited for this study. The copy numbers of ΔmtDNA⁴⁹⁷⁷ and wild-type mtDNA were determined by real-time quantitative PCR and analyzed using Spearman’s bivariate correlation analysis, t-tests, or one-way ANOVA. The overall ΔmtDNA⁴⁹⁷⁷ copy number per cell and the proportion of mtDNA⁴⁹⁷⁷ relative to the total wild-type mtDNA, increased with patient age and symptom severity. Surprisingly, the total mtDNA copy number decreased with increasing symptom severity. Our analyses revealed that increases in the proportion and total copy number of ΔmtDNA⁴⁹⁷⁷ in the blood may be associated with disease severity in patients with mitochondrial dysfunction.

The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.