Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

BRCA1 Intragenic Duplication Combined with a Likely Pathogenic TP53 Variant in a Patient with Triple-Negative Breast Cancer: Clinical Risk and Management.

For patients with hereditary breast and ovarian cancer, the probability of carrying two pathogenic variants (PVs) in dominant cancer-predisposing genes is rare. Using targeted next-generation sequencing (NGS), we investigated a 49-year-old Caucasian woman who developed a highly aggressive breast tumor. Our analyses identified an intragenic germline heterozygous duplication in BRCA1 with an additional likely PV in the TP53 gene. The BRCA1 variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints were characterized at the nucleotide level (c.135-2578_442-1104dup). mRNA extracted from lymphocytes was amplified by RT-PCR and then Sanger sequenced, revealing a tandem duplication r.135_441dup; p.(Gln148Ilefs*20). This duplication results in the synthesis of a truncated and, most likely, nonfunctional protein. Following functional studies, the TP53 exon 5 c.472C > T; p.(Arg158Cys) missense variant was classified as likely pathogenic by the Li-Fraumeni Syndrome (LFS) working group. This type of unexpected association will be increasingly identified in the future, with the switch from targeted BRCA sequencing to hereditary breast and ovarian cancer (HBOC) panel sequencing, raising the question of how these patients should be managed. It is therefore important to record and investigate these rare double-heterozygous genotypes.

A missense mutation in TTC8/BBS8 affecting mRNA splicing in patients with non-syndromic retinitis pigmentosa.

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.

Identification and Characterization of an Exonic Duplication in PALB2 in a Man with Synchronous Breast and Prostate Cancer.

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

Fabry disease: Detection of Alu-mediated exon duplication by NGS.

Monogenetic diseases can be analyzed routinely by targeted DNA sequencing. If causative variants are not found, complementary methods like RNA sequencing or analysis of copy number variations by multiplex ligation-dependent probe amplification have to be considered. In the latter, especially exonic duplications or deletions can be detected, but the precise sites of mutations remain unclear. As we demonstrate in this casuistic report of Fabry disease, next-generation sequencing (NGS) of a long-range PCR product can identify the recombination site directly and illuminate the underlying molecular mechanism.

A chelicerate-specific burst of nonclassical Dscam diversity.

BACKGROUND: The immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals. RESULTS: In this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types-mDscam, sDscamα, sDscamß, and sDscamγ-based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5' cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams. CONCLUSIONS: This study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire.

Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis.

TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.

SRD5A3-CDG: Twins with an intragenic tandem duplication.

Steroid 5α-reductase type 3 congenital disorder of glycosylation (SRD5A3-CDG) is a rare metabolic disease mainly characterized by psychomotor disability, visual impairment, and variable eye malformations caused by bi-allelic pathogenic variants in SRD5A3. So far, only 23 distinct mutations were described. Exome sequencing in 32-year old monozygotic male twins revealed only the heterozygous splice variant c.562+3delG in SRD5A3, but no second variant. The twins presented with psychomotor deficit and a complex eye disease including retinal dystrophy, pallor of the papilla, nystagmus, and strabismus suggestive of SRD5A3-CDG. Only when applying exome-based copy number analysis, we identified as a second compound heterozygous variant a previously not reported tandem duplication of exons 2-4 in SRD5A3. Next to the typical skeletal anomalies of SRD5A3-CDG such as kyphosis and scoliosis, extension deficits of the proximal interphalangeal (PIP) joints IV were observed. Since similar contractures were described once in a patient with SRD5A3-CDG, we suggest that this rare symptom is possibly associated with SRD5A3-CDG. Our findings further expand the mutational and clinical spectrum of SRD5A3-CDG and emphasize the importance of an intragenic copy number analysis in patients with strong clinical suspicion of SRD5A3-CDG and only one detectable sequence variant.

Origins and Evolution of Human Tandem Duplicated Exon Substitution Events.

The mutually exclusive splicing of tandem duplicated exons produces protein isoforms that are identical save for a homologous region that allows for the fine tuning of protein function. Tandem duplicated exon substitution events are rare, yet highly important alternative splicing events. Most events are ancient, their isoforms are highly expressed, and they have significantly more pathogenic mutations than other splice events. Here, we analyzed the physicochemical properties and functional roles of the homologous polypeptide regions produced by the 236 tandem duplicated exon substitutions annotated in the human gene set. We find that the most important structural and functional residues in these homologous regions are maintained, and that most changes are conservative rather than drastic. Three quarters of the isoforms produced from tandem duplicated exon substitution events are tissue-specific, particularly in nervous and cardiac tissues, and tandem duplicated exon substitution events are enriched in functional terms related to structures in the brain and skeletal muscle. We find considerable evidence for the convergent evolution of tandem duplicated exon substitution events in vertebrates, arthropods, and nematodes. Twelve human gene families have orthologues with tandem duplicated exon substitution events in both Drosophila melanogaster and Caenorhabditis elegans. Six of these gene families are ion transporters, suggesting that tandem exon duplication in genes that control the flow of ions into the cell has an adaptive benefit. The ancient origins, the strong indications of tissue-specific functions, and the evidence of convergent evolution suggest that these events may have played important roles in the evolution of animal tissues and organs.

Evolutionary Genetic Signatures of Selection on Bone-Related Variation within Human and Chimpanzee Populations.

Bone strength and the incidence and severity of skeletal disorders vary significantly among human populations, due in part to underlying genetic differentiation. While clinical models predict that this variation is largely deleterious, natural population variation unrelated to disease can go unnoticed, altering our perception of how natural selection has shaped bone morphologies over deep and recent time periods. Here, we conduct the first comparative population-based genetic analysis of the main bone structural protein gene, collagen type I α 1 (COL1A1), in clinical and 1000 Genomes Project datasets in humans, and in natural populations of chimpanzees. Contrary to predictions from clinical studies, we reveal abundant COL1A1 amino acid variation, predicted to have little association with disease in the natural population. We also find signatures of positive selection associated with intron haplotype structure, linkage disequilibrium, and population differentiation in regions of known gene expression regulation in humans and chimpanzees. These results recall how recent and deep evolutionary regimes can be linked, in that bone morphology differences that developed among vertebrates over 450 million years of evolution are the result of positive selection on subtle type I collagen functional variation segregating within populations over time.