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Regions of homozygosity (ROH) could implicate uniparental disomy (UPD) on specific chromosomes associated with imprinting disorders. Though the algorithms for ROH detection in exome sequencing (ES) have been developed, optimal reporting thresholds and when to pursue confirmatory UPD testing for imprinting disorders remain in ambiguity. This study used a data-driven approach to assess optimal reporting thresholds of ROH in clinical practice. ROH analysis was performed using Automap in a retrospective cohort of 8,219 patients and a prospective cohort of 1,964 patients with ES data. Cases with ROH on imprinting-disorders related chromosomes were selected for additional methylation-specific confirmatory testing. The diagnostic yield, the ROH pattern of eventually diagnosed cases and optimal thresholds for confirmatory testing were analyzed. In the retrospective analysis, 15 true UPD cases of imprinting disorders were confirmed among 51 suspected cases by ROH detection. Pattern of ROH differed between confirmed UPD and non-UPD cases. Maximized yield and minimized false discovery rate of confirmatory UPD testing was achieved at the thresholds of >20 Mb or >25 % chromosomal coverage for interstitial ROH, and >5 Mb for terminal ROH. Current recommendation by ACMG was nearly optimal, though refined thresholds as proposed in this study could reduce the workload by 31 % without losing any true UPD diagnosis. Our refined thresholds remained optimal after independent evaluation in a prospective cohort. ROH identified in ES could implicate the presence of clinically relevant UPD. This study recommended size and coverage thresholds for confirmatory UPD testing after ROH detection in ES, contributing to the development of evidence-based reporting guidelines. [ABSTRACT FROM AUTHOR]

Achondroplasia and hypochondroplasia are the most common forms of disproportionate short stature, of which the vast majority of cases can be attributed to the hotspot missense mutations in the gene FGFR3. Here we presented cases with a novel cryptic splicing variant of FGFR3 gene and aimed to interrogate the variant pathogenicity. In whole exome sequencing of two patients with hypochondroplasia-like features, a de novo intronic variant c.1075 + 95C>G was identified, predicted to alter mRNA splicing. Minigene assay showed that this intronic variant caused retention of a 90-nucleotide segment of intron 8 in mRNA, resulting in a 30-amino acid insertion at the extracellular domain of the protein. This is the first likely pathogenic splicing variant identified in the FGFR3 gene and was detected in one additional patient among 26 genetically unresolved patients. Our results strongly suggest that c.1075 + 95C>G is a recurrent mutation and should be included in genetic testing of FGFR3 especially for those patients with equivocal clinical findings and no exonic mutations identified. [ABSTRACT FROM AUTHOR]

Objectives: Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis.11β-hydroxylase deficiency and 17α-hydroxylase deficiency are two forms of CAH caused by defects of CYP11B1 and CYP17A1 respectively. Case presentation: Two rare intronic variants were identified in suspected CAH patients. Though not located at the classic splicing sites, these two variants perturbed splicing based on minigene assays. One variant, NM_000497.4: c.240-157T>G of CYP11B1 identified in subject 1, resulted in the retention of 136 intronic nucleotides. The other variant, NM_000102.4: c.754-6 A>G of CYP17A1 identified in subject 2, leading to the retention of 5 intronic nucleotides. Both variants resulted in out-of-frame alteration of the respective transcript. Conclusion: Cryptic splicing variants in the intronic regions contribute to the genetic defects of CAH. Minigene assay is useful to confirm the splice altering effect and make a definitive molecular diagnosis. [ABSTRACT FROM AUTHOR]

Background: Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods: Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results: The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions: The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes. [ABSTRACT FROM AUTHOR]

Background: This study aimed to identify the predictive value of basal sex hormone levels for activation of the hypothalamic–pituitary–gonadal (HPG) axis in girls. Methods: Gonadotropin-releasing hormone (GnRH) stimulation tests were performed and evaluated in a total of 1750 girls with development of secondary sex characteristics. Correlation analyses were conducted between basal sex hormones and peak luteinizing hormone (LH) levels ≥5 IU/L during the GnRH stimulation test. Receiver operating characteristic (ROC) curves for basal levels of LH, follicle-stimulating hormone (FSH), LH/FSH, and estradiol (E2) before the GnRH stimulation test were plotted. The area under the curve (AUC) and 95% confidence intervals (CIs) were measured for each curve. Results: The maximum AUC value was observed for basal LH levels (0.77, 95% CI: 0.74–0.79), followed by basal FSH levels (0.73, 95% CI: 0.70–0.75), the basal LH/FSH ratio (0.68, 95% CI: 0.65–0.71), and basal E2 levels (0.61, 95% CI: 0.59–0.64). The appropriate cutoff value of basal LH levels associated with a positive response of the GnRH stimulation test was 0.35 IU/L, with a sensitivity of 63.96% and specificity of 76.3% from the ROC curves when Youden’s index showed the maximum value. When 100% of patients had peak LH levels ≥5 IU/L, basal LH values were >2.72 IU/L, but the specificity was only 5.45%. Conclusions: Increased basal LH levels are a significant predictor of a positive response during the GnRH stimulation test for assessing activation of the HPG axis in most girls with early pubertal signs. [ABSTRACT FROM AUTHOR]

Familial hypercholesterolemia (FH) is an autosomal dominant, inherited disease (OMIM 143890) characterized by elevated serum cholesterol bound to low-density lipoprotein (LDL). It is mainly caused by mutations of the low-density lipoprotein receptor gene ( LDLR). In this study, we investigated two Chinese pedigrees with FH. The probands were a 9-year-old boy and a 1-year-old boy, who had high LDL-C levels. The proband in family A showed skin xanthoma. We sequenced the promoter and all exons and exon-intron boundaries of the LDLR gene to detect potential mutations. Compound heterozygote of c.1747C>T and c.2054C>T was detected in the proband of family A, and a heterozygous indel mutation c.551_553 delGTAinsTT was discovered in the second family. The c.1747C>T and c.2054C>T mutations, which have been reported previously, result in His583Tyr and Pro685Leu substitutions, respectively. The novel c.551_553 delGTAinsTT indel mutation causes a frameshift, which results in a p.Cys184Phe fs21X mutation in the corresponding protein. [ABSTRACT FROM AUTHOR]