A Novel Homozygous PKP2 Variant in Severe Neonatal Non-compaction and Concomitant Ventricular Septal Defect: A Case Report

Left ventricular non-compaction (LVNC) is a rare and genetically heterogeneous cardiomyopathy. The disorder vastly affects infants and young children. Severe neonatal LVNC is relatively rare. The prevalence of genetic defects underlying pediatric and adult-onset LVNC is about 17–40%. Mutations of MYH7 and MYBPC3 sarcomeric genes are found in the vast majority of the positive pediatric cases. PKP2 encodes plakophilin-2, a non-sarcomeric desmosomal protein, which has multiple roles in cardiac myocytes including cell–cell adhesion, tightening gap junction, and transcriptional factor. Most of the reported PKP2 mutations are heterozygous missense and truncating variants, and they are associated with an adult-onset autosomal dominant disorder, namely arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). Homozygous PKP2 mutations have been rarely described. Herein, we present a rare case of an infant with neonatal onset of congestive heart failure owing to severe LVNC and multiple muscular VSD. Medical treatments failed to control the heart failure and the patient died at 11 months of age. Whole-exome sequencing identified a novel homozygous PKP2 variant, c.1511-1G>C, in the patient. An mRNA analysis revealed aberrant transcript lacking exon 7, which was predicted to cause a frameshift and truncated peptide (p.Gly460GlufsTer2). The heterozygous parents had normal cardiac structures and functions as demonstrated by electrocardiogram and echocardiography. Pathogenic variants of sarcomeric genes analyzed were not found in the patient. We conducted a literature review and identified eight families with biallelic PKP2 mutations. We observed that three families (our included) with null variants were linked to lethal phenotypes, while homozygous missense mutations resulted in less severe manifestations: adolescent-onset ARVD/C and childhood-onset DCM. Our data support a previous notion that severe neonatal LVNC might represent a unique entity and had distinct genetic spectrum. In conclusion, the present study has extended the phenotypes and genotypes of PKP2-related disorders and lethal LVNC.

RNA assay identifies a previous misclassification of BARD1 c.1977A>G variant

Case–control studies have shown an association of BARD1 with hereditary breast and/or ovarian cancer (HBOC) predisposition. BARD1 alternatively spliced isoforms are abundant and some are highly expressed in different cancer types. In addition, a number of BARD1 germline pathogenic variants have been reported among HBOC patients. In previous reports, BARD1 c.1977A>G variant has been classified as pathogenic since it produces a frameshift transcript lacking exons 2 to 9. In the present study, we sought to validate the mRNA splicing results previously published and to contribute with new evidence to refine the classification of this substitution according to ACMG/AMP guidelines. The presence of the variant was screened in patients and controls. RT-PCR was performed in order to compare the transcriptional profiles of two variant carriers and ten non-carrier controls. In addition, allele-specific expression was assessed. No differences in variant frequency were detected between patients and controls. The RNA assay confirmed the presence of the shorter transcript lacking exons 2–9, but it was detected both in carriers and non-carriers. Furthermore, allelic imbalance was discarded and no significant differences in the proportion of full-length and shorter transcript were detected between carriers and controls. The shorter transcript detected corresponds to BARD1 isoform η, constituted by exons 1, 10 and 11. Our results support that this transcript is a constitutive splicing product rather than an aberrant transcript caused by BARD1 c.1977A>G variant, and for this reason this variant should be considered as likely benign following ACMG/AMP guidelines.

Classification of the canonical splice alteration MUTYH c.934-2A>G is likely benign based on RNA and clinical data

MUTYH-associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in MUTYH are responsible for MAP. The MUTYH c.934-2A>G (NM_001128425.1) variant, which is also known as c.850-2A>G for NM_001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) due to lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nucleotides at the start of exon 11 (MUTYH r.934_942del9). This event is predicted to lead to an in-frame loss of 3 amino acids in a non-critical domain of the protein. This was the only splice defect identified in these patients which was not present in the controls and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, MUTYH c.934-2A>G. The splicing analysis demonstrating a small in-frame skipping of 3 amino acids in a non-critical domain along with the absence of a MAP phenotype in our internal cohort of biallelic carriers provides evidence that the variant is likely benign and not of clinical significance.

Aberrant transcript usage induces homologous recombination deficiency and predicts therapeutic responses

BRCA1/2 mutations account for only a small fraction of homologous recombination (HR) deficiency (HRD) cases. Recently developed genomic HRD (gHRD) tests suffer confounding factors causing low precision in predicting samples that will respond to poly (ADP-ribose) polymerase (PARP) inhibitors and DNA damaging agents. Here, we present molecular evidence and clinical utility of transcriptional HRD (tHRD) that is based on aberrant transcript usage (TU) of minor isoforms. Specifically, increased TU of non-functional isoforms of DNA repair genes was prevalent in breast and ovarian cancer with gHRD. Our functional assays validated its association with impaired HR activity. Remarkably, tHRD detection based on the TU pattern of key genes was superior to gHRD or BRCA1/2 screening in accuracy for predicting the responses of cell lines and cancer patients to PARP inhibitors and genotoxic drugs. In particular, this approach demonstrated the capability to reflect functional HR status, particularly when applied to our cohort of olaparib users with acquired platinum resistance in ovarian cancer. Hence, the tHRD-based diagnostic tests are expected to broaden the clinical utility of PARP inhibitors.

Identification of SOFT syndrome caused by a pathogenic homozygous splicing variant of POC1A: a case report

Background Pathogenic variants in POC1A led to SOFT syndrome and variant POC1A-related (vPOC1A) syndrome. SOFT syndrome is a rare primordial dwarfism condition characterized by short stature, onychodysplasia, facial dysmorphism and hypotrichosis.The main clinical differences between SOFT and vPOC1A syndrome include dyslipidemia with insulin resistance and acanthosis nigricans. To our knowledge, this is the first report of a SOFT syndrome patient diagnosed with a homozygous splicing variant, which could help to extend our understanding of the genotypic and phenotypic information of the disease. Case presentation We reported a seven-year-old boy with SOFT syndrome. The patient presented symmetrical short stature and facial features, including prominent forehead, inverted triangular face, epicanthal fold, small teeth and enlarged ears. Laboratory tests displayed mild insulin resistance. Whole-exome sequencing (WES) led to the identification of a homozygous splicing variant (c.981+1G>A) in POC1A gene of the patient, which was inherited from his heterozygous parents confirmed by Sanger sequencing. Further transcriptional experiments of the splicing variant revealed aberrant percentage of exon 9 skipping transcripts. Conclusions This is the firstly reported case of a SOFT syndrome patient with a novel homozygous splicing variant and detailed delineation of the aberrant transcript in proband and carrier of the variant in Chinese. Our study enriched mutational spectrum of POC1A which could help in further genetic diagnosis and counselling of SOFT syndrome patients.

Novel deep intronic mutation in PLA2G6 causing early-onset Parkinson’s disease with brain iron accumulation through pseudo-exon activation

PLA2G6 is the causative gene for a group of autosomal recessive neurodegenerative disorders known as PLA2G6-associated neurodegeneration (PLAN). We present a case with early-onset parkinsonism, ataxia, cognitive decline, cerebellar atrophy, and brain iron accumulation. Sequencing of PLA2G6 coding regions identified only a heterozygous nonsense variant, but mRNA analysis revealed the presence of an aberrant transcript isoform due to a novel deep intronic variant (c.2035-274G > A) leading to activation of an intronic pseudo-exon. These results expand the genotypic spectrum of PLAN, showing the paramount importance of detecting possible pathogenic variants in deep intronic regions in undiagnosed patients.

SPLICE-q: a Python tool for genome-wide quantification of splicing efficiency

Background Introns are generally removed from primary transcripts to form mature RNA molecules in a post-transcriptional process called splicing. An efficient splicing of primary transcripts is an essential step in gene expression and its misregulation is related to numerous human diseases. Thus, to better understand the dynamics of this process and the perturbations that might be caused by aberrant transcript processing it is important to quantify splicing efficiency. Results Here, we introduce SPLICE-q, a fast and user-friendly Python tool for genome-wide SPLICing Efficiency quantification. It supports studies focusing on the implications of splicing efficiency in transcript processing dynamics. SPLICE-q uses aligned reads from strand-specific RNA-seq to quantify splicing efficiency for each intron individually and allows the user to select different levels of restrictiveness concerning the introns’ overlap with other genomic elements such as exons of other genes. We applied SPLICE-q to globally assess the dynamics of intron excision in yeast and human nascent RNA-seq. We also show its application using total RNA-seq from a patient-matched prostate cancer sample. Conclusions Our analyses illustrate that SPLICE-q is suitable to detect a progressive increase of splicing efficiency throughout a time course of nascent RNA-seq and it might be useful when it comes to understanding cancer progression beyond mere gene expression levels. SPLICE-q is available at: https://github.com/vrmelo/SPLICE-q

Identification of a Splice Variant (c.5074+3A>C) of BRCA1 by RNA Sequencing and TOPO Cloning

Grading the pathogenicity of BRCA1/2 variants has great clinical importance in patient treatment as well as in the prevention and screening of hereditary breast and ovarian cancer (HBOC). For accurate evaluation, confirming the splicing effect of a possible splice site variant is crucial. We report a significant splicing variant (c.5074+3A>C) in BRCA1 in a patient with recurrent ovarian cancer. Next-generation sequencing (NGS) of BRCA1/2 from patient’s peripheral blood identified the variant, which was strongly suspected of being a splicing mutation based on in silico predictions. Direct RNA analysis yielded multiple transcripts, and TOPO cloning of the complementary DNA (cDNA) and Sanger sequencing revealed an aberrant transcript with an insertion of the first 153 bp of intron 17, and another transcript with the 153 bp insertion along with an exon 18 deletion. A premature termination codon was presumed to be formed by the 153 bp partial intron retention common to the two transcripts. Therefore, BRCA1 c.5074+3A>C was classified as a likely pathogenic variant. Our findings show that active use of functional studies of variants suspected of altered splicing are of great help in classifying them.

Allelic and phenotypic heterogeneity in Junctophillin-3 related neurodevelopmental and movement disorders

Junctophilin-3 belongs to a triprotein junctional complex implicated in the regulation of neuronal excitability and involved in the formation of junctional membrane structures between voltage-gated ion channels and endoplasmic (ryanodine) reticular receptors. A monoallelic trinucleotide repeat expansion located within the junctophilin-3 gene (JPH3) has been implicated in a rare autosomal dominant (AD) late-onset (and progressive) disorder clinically resembling Huntington disease (HD), and known as HD-like 2 (HDL2; MIM# 606438). Although the exact molecular mechanisms underlying HDL2 has not yet been fully elucidated, toxic gain-of-function of the aberrant transcript (containing the trinucleotide repeat) and loss of expression of (full-length) junctophilin-3 have both been implicated in HDL2 pathophysiology. In this study, we identified by whole exome sequencing (WES) a JPH3 homozygous truncating variant [NM_020655.4: c.17405dup; p.(Val581Argfs*137)]. in a female individual affected with genetically undetermined neurodevelopmental anomalies (including delayed motor milestones, abnormal social communication, language difficulties and borderline cognitive impairment) and paroxysmal attacks of dystonia since her early infancy. Our study expands the JPH3-associated mutational spectrum and clinical phenotypes, implicating the loss of Junctophilin-3 in heterogeneous neurodevelopmental phenotypes and early-onset paroxysmal movement disorders.