Characterization of Synonymous BRCA1:c.132C>T as a Pathogenic Variant

Breast cancer gene 1 (BRCA1) and BRCA2 are tumor suppressors involved in DNA damage response and repair. Carriers of germline pathogenic or likely pathogenic variants in BRCA1 or BRCA2 have significantly increased lifetime risks of breast cancer, ovarian cancer, and other cancer types; this phenomenon is known as hereditary breast and ovarian cancer (HBOC) syndrome. Accurate interpretation of BRCA1 and BRCA2 variants is important not only for disease management in patients, but also for determining preventative measures for their families. BRCA1:c.132C>T (p.Cys44=) is a synonymous variant recorded in the ClinVar database with “conflicting interpretations of its pathogenicity”. Here, we report our clinical tests in which we identified this variant in two unrelated patients, both of whom developed breast cancer at an early age with ovarian presentation a few years later and had a family history of relevant cancers. Minigene assay showed that this change caused a four-nucleotide loss at the end of exon 3, resulting in a truncated p.Cys44Tyrfs*5 protein. Reverse transcription-polymerase chain reaction identified two fragments (123 and 119 bp) using RNA isolated from patient blood samples, in consistency with the results of the minigene assay. Collectively, we classified BRCA1:c.132C>T (p.Cys44=) as a pathogenic variant, as evidenced by functional studies, RNA analysis, and the patients’ family histories. By analyzing variants recorded in the BRCA Exchange database, we found synonymous changes at the ends of exons could potentially influence splicing; meanwhile, current in silico tools could not predict splicing changes efficiently if the variants were in the middle of an exon, or in the deep intron region. Future studies should attempt to identify variants that influence gene expression and post-transcription modifications to improve our understanding of BRCA1 and BRCA2, as well as their related cancers.

Single-nucleotide polymorphisms in <i>FLT3</i>, <i>NLRP5</i>, and <i>TGIF1</i> are associated with litter size in Small-tailed Han sheep

Previous studies have indicated that FLT3, NLRP5, and TGIF1 play a pivotal role in sheep fecundity. Nevertheless, little is known about the association of the polymorphisms of these genes with litter size (LS). In this study, the selected single-nucleotide polymorphisms (SNPs) were genotyped using a Sequenom MassARRAY® platform, and the distribution of different genotypes of the SNPs in the seven sheep breeds (Small-tailed Han, Hu, Cele Black, Suffolk, Tan, Prairie Tibetan, and Sunite sheep) were analyzed. The reliability of the estimated allele frequency for all seven SNPs was at least 0.9545. Given the association of the TGIF1 g.37866222C > T polymorphism with LS in Small-tailed Han sheep (p<0.05), fecundity differences might be caused by the change in amino acid from proline (Pro) to serine (Ser), which has an impact on secondary, tertiary protein structures with concomitant TGIF1 functionality changes. The FLT3 rs421947730 locus has a great effect on the LS (p<0.05), indicating that the locus of FLT3 in synergy with KILTG is likely to facilitate ovarian follicle maturation and ovulation. Moreover, NLRP5 rs426897754 is associated with the LS of the second and third parities (p<0.05). We speculate that a synonymous variant of NLRP5 may be involved in folliculogenesis accompanied by BMP15, FSHR, BMPR1B, AMH, and GDF9, resulting in the different fecundity of Small-tailed Han sheep. Our studies provide valuable genetic markers for sheep breeding.

Evaluation for Retinal Therapy for RPE65 Variation Assessed in hiPSC Retinal Pigment Epithelial Cells

Human induced pluripotent stem cells (hiPSCs) generated from patients and the derivative retinal cells enable the investigation of pathological and novel variants in relevant cell populations. Biallelic pathogenic variants in RPE65 cause early-onset severe retinal dystrophy (EOSRD) or Leber congenital amaurosis (LCA). Increasingly, regulatory-approved in vivo RPE65 retinal gene replacement therapy is available for patients with these clinical features, but only if they have biallelic pathological variants and sufficient viable retinal cells. In our cohort of patients, we identified siblings with early-onset severe retinal degeneration where genomic studies revealed compound heterozygous variants in RPE65, one a known pathogenic missense variant and the other a novel synonymous variant of uncertain significance. The synonymous variant was suspected to affect RNA splicing. Since RPE65 is very poorly expressed in all tissues except the retinal pigment epithelium (RPE), we generated hiPSC-derived RPE cells from the parental carrier of the synonymous variant. Sequencing of RNA obtained from hiPSC-RPE cells demonstrated heterozygous skipping of RPE65 exon 2 and the introduction of a premature stop codon in the mRNA. Minigene studies confirmed the splicing aberration. Results from this study led to reclassification of the synonymous variant to a pathogenic variant, providing the affected patients with access to RPE65 gene replacement therapy.

Bi-Allelic c.1746G>T; p.Leu582= Variants in TUBGCP4 in a Boy with Autism: Clinical Data and Literature Review

Bi-allelic mutations in the <i>TUBGCP4</i> gene have been recently associated with autosomal recessive microcephaly with chorioretinopathy. However, little is known about the genotype-phenotype characteristics of this disorder. Here, we describe a 5-year-old male patient with autism and a normal occipitofrontal circumference. No retinal abnormalities were observed. Brain MRI revealed the presence of enlarged sheaths of both tortuous optic nerves; both eyes had shorter axial lengths. Whole-exome sequencing in trio revealed synonymous <i>TUBGCP4</i> variants in homozygous state: c.1746G&#x3e;T; p.Leu582=. This synonymous variant has been previously described and probably leads to skipping of exon 16 of <i>TUBGCP4</i>. These results broaden the clinical spectrum of this new syndrome and suggest that <i>TUBGCP4</i> bi-allelic mutations may underlie complex neurodevelopmental disorders.

A novel synonymous ABCA3 variant identified in a Chinese family with lethal neonatal respiratory failure

Background Lethal respiratory failure is primarily caused by a deficiency of pulmonary surfactant, and is the main cause of neonatal death among preterm infants. Pulmonary surfactant metabolism dysfunction caused by variants in the ABCA3 gene is a rare disease with very poor prognosis. Currently, the mechanisms associated with some ABCA3 variants have been determined, including protein mistrafficking and impaired phospholipid transport. However, some novel variants and their underlying pathogenesis has not been fully elucidated yet. In this study we aimed to identify the genetic features in a family with lethal respiratory failure. Methods We studied members of two generations of a Chinese family, including a female proband, her parents, her monozygotic twin sister, and her older sister. Trio whole exome sequencing (WES) were used on the proband and her parents to identify the ABCA3 variants. Sanger sequencing and real-time quantitative polymerase chain reaction (PCR) were used on the monozygotic twin sister of proband to validate the ABCA3 synonymous variant and exon deletion, respectively. The potential pathogenicity of the identified synonymous variant was predicted using the splice site algorithms dbscSNV11_AdaBoost, dbscSNV11_RandomForest, and Human Splicing Finder (HSF). Results All patients showed severe respiratory distress, which could not be relieved by mechanical ventilation, supplementation of surfactant, or steroid therapy, and died at an early age. WES analysis revealed that the proband had compound heterozygous ABCA3 variants, including a novel synonymous variant c.G873A (p.Lys291Lys) in exon 8 inherited from the mother, and a heterozygous deletion of exons 4–7 inherited from the father. The synonymous variant was consistently predicted to be a cryptic splice donor site that may lead to aberrant splicing of the pre-mRNA by three different splice site algorithms. The deletion of exons 4–7 of the ABCA3 gene was determined to be a likely pathogenic variant. The variants were confirmed in the monozygotic twin sister of proband by Sanger sequencing and qPCR respectively. The older sister of proband was not available to determine if she also carried both ABCA3 variants, but it is highly likely based on her clinical course. Conclusions We identified a novel synonymous variant and a deletion in the ABCA3 gene that may be responsible for the pathogenesis in patients in this family. These results add to the known mutational spectrum of the ABCA3 gene. The study of ABCA3 variants may be helpful for the implementation of patient-specific therapies.

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus

Aspergillus fumigatus is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant A. fumigatus strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences—comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP–SNP interactions impacting progeny growth at different AMB concentrations.

Analysis of mutations associated with Parkinson’s disease in patients of the Krasnoyarsk region

Background. Parkinson’s disease (PD) is one of the common neurodegenerative diseases. Several genes are known (SNCA, PARK2, PINK1, PARK7 (DJ-1) and LRRK2), mutations in which have a pathological significance in the development of monogenic PD; association with PD of other genes (for example, UCHL1, ATP13A2) requires further study. It is also known, that GBA gene is associated with an increased risk of PD developing.Aim. Analysis of mutations and polymorphisms in the PARK2, PINK1, SNCA, ATP13A2, PARK7, LRRK2, UCHL, GCH1 and GBA genes in patients with PD from Krasnoyarsk region.Material and methods. The 60 patients with sporadic and familial forms of PD were included in the study. The SALSA MLPA Holland P051 and P052 kits («MRC Amsterdam», The Netherlands) were used to detect deletions and duplications in the PARK2, PINK1, SNCA, ATP13A2, PARK7, LRRK2, UCHL, GCH1 genes, as well as point mutations A30P in the SNCA gene and G2019S in the LRRK2 gene. Analysis of the GBA gene was carried out by Senger sequencing.Results. None of the 60 patients had mutations that were searched with the SALSA MLPA Holland P051 and P052 kits. 6 different mutations in the GBA gene were found in 9 out of 60 patients with PD. L444P («severe» PD — associated mutation) — in two patients, D409H («severe» PD — associated mutation) — in one patient, T369M (polymorphism, possibly associated with PD) — in two patients, E326K (polymorphism, possibly associated with PD) — in one patient, V460V (synonymous variant, which is part of the composition of the complex RecNcil mutation (p.L444P; p.A456P; p.V460V) associated with PD) — in two patients and variant C.*92g>A (3’ — UTR polymorphism, possibly associated with PD) — in one patient. Two patients had compound heterozygous carriers of two variants.Conclusion. This paper presents the genetic analysis results of the PD associated genes among patients from the Krasnoyarsk region. No mutations were detected in the PARK2, PINK1, SNCA, ATP13A2, PARK7, LRRK2, UCHL and GCH1 genes. Genetic variants analysis of the GBA gene showed similar frequency in the patients from the Krasnoyarsk region as in European populations.