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RNA sequencing (RNA-seq) is a complementary diagnostic tool to exome sequencing (ES), only recently clinically available to undiagnosed patients post-ES, that provides functional information on variants of unknown significance (VUS) by evaluating its effect on RNA transcription. ES became clinically available in the early 2010s and promised an agnostic platform for patients with a neurological disease, especially for those who believed to have a genetic etiology. However, the massive data generated by ES pose challenges in variant interpretation, especially for rare missense, synonymous, and deep intronic variants that may have a splicing effect. Without functional study and/or family segregation analysis, these rare variants would be likely interpreted as VUS which is difficult for clinicians to use in clinical care. Clinicians are able to assess the VUS for phenotypic overlap, but this additional information alone is usually not enough to re-classify a variant. Here, we report a case of a 14-month-old male who presented to clinic with a history of seizures, nystagmus, cerebral palsy, oral aversion, global developmental delay, and poor weight gain requiring gastric tube placement. ES revealed a previously unreported homozygous missense VUS, c.7406A > G p.(Asn2469Ser), in VPS13D. This variant has not been previously reported in genome aggregation database (gnomAD), ClinVar, or in any peer-reviewed published literature. By RNA-seq, we demonstrated that this variant mainly impacts splicing and results in a frameshift and early termination. It is expected to generate either a truncated protein, p.(Val2468fs*19), or no protein from this transcript due to nonsense-mediated mRNA decay leading to VPS13D deficiency. To our knowledge, this is the first case utilizing RNA-seq to further functionally characterize a homozygous novel missense VUS in VPS13D and confirm its impact on splicing. This confirmed pathogenicity gave the diagnosis of VPS13D movement disorder to this patient. Therefore, clinicians should consider utilizing RNA-seq to clarify VUS by evaluating its effect on RNA transcription.

Upshaw-Schulman syndrome (USS)-rare autosomal recessive disease that affects <1/1 000 000 individuals. It is characterized by the massive formation of platelet thrombi in the microcirculation accompanied by haemolytic anaemia, thrombocytopenia and clinical and laboratory signs of renal and neurological failure. USS is caused by mutations in the ADAMTS13 gene. Mutations in the ADAM metallopeptidase with thrombospondin type 1 motif 13  (ADAMTS13) gene can lead to disruption of secretion of this enzyme, or to decrease of enzyme proteinase activity without effect on ADAMTS13 secretion. The aim of this work is to describe a clinical case of USS caused by a new missense mutation in the ADAMTS13 gene. The diagnosis of thrombotic thrombocytopenic purpura was based on clinical signs and confirmed if plasma ADAMTS13 activity was <10%. ADAMTS13 gene sequencing was performed by the Sanger method using oligonucleotide primers of our own design. We found a new, undescribed mutation p.Trp387Ser in a TrpXXTrp motif. Previously, a pathogenic variation disrupting the 387TrpSerSerTrp390 motif of the ADAMTS13 protein was detected only once. Clinical picture of a patient with the combination of the p.Trp387Ser and p.Arg1060Trp variations is quite similar to that of the homozygous state of p.Arg1060Trp variant.

Multiple non-related neoplasia does not have an established approach or benefits for performing whole-exome sequencing (WES) analysis. We report on a 46-year-old woman who developed astrocytoma, thyroid, and breast cancer within 10 years. The WES analysis found a novel missense variant in the ACSL6 gene, and the protein modeling showed altered secondary and tertiary structures, which modify the binding to cofactors and substrates. ACSL6 is involved in lipid metabolism, expressed in the brain, thyroid, and breast tissues, and is associated with diverse types of cancer. Our study demonstrates the benefit of WES analysis compared with commercial panels in patients with non-related neoplasia.

Variants in HCN1 are associated with a range of epilepsy syndromes including developmental and epileptic encephalopathies. Here we describe a child harboring a novel de novo HCN1 variant, E246A, in a child with epilepsy and mild developmental delay. By parental report, the child had difficulty in discriminating between colors implicating a visual deficit. This interesting observation may relate to the high expression of HCN1 channels in rod and cone photoreceptors where they play an integral role in shaping the light response. Functional analysis of the HCN1 E246A variant revealed a right shift in the voltage dependence of activation and slowing of the rates of activation and deactivation. The changes in the biophysical properties are consistent with a gain-of-function supporting the role of HCN1 E246A in disease causation. This case suggests that visual function, including color discrimination, should be carefully monitored in patients with diseases due to HCN1 pathogenic variants.

For genes with reliable estimates of disease risk associated with loss-of-function variants, case-control data can be used to estimate the proportion of variants of typical risk effect for defined groups of variants, of relevance for variant classification. A calculation was derived for a maximum likelihood estimate of the proportion of pathogenic variants of typical effect from case-control data and applied to rare variant counts for ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, and RAD51D from published breast cancer studies: BEACCON (5770 familial cases and 5741 controls) and breast cancer risk after diagnostic sequencing (60,466 familial and population-based cases and 53,461 controls). There was significant evidence of pathogenic variants among rare noncoding variants, in particular deeper intronic variants, for BRCA1 (13%, p = 8.3 × 10-7 ), BRCA2 (6%, p = 0.016) and PALB2 (13%, p = 0.001). The estimated proportion of pathogenic missense variants varied markedly between genes, generally with enrichment in familial cases, for example, 9% for BRCA2 versus 60%-90% for CHEK2. Stratifying missense variants by position indicated that, for most genes, location within a functional domain significantly predicted pathogenicity, whereas location outside domains provided robust evidence against pathogenicity. Our approach provides novel insights into the spectrum of pathogenic variants of specific breast cancer genes and has wider application to inform gene-focused specifications of American College of Medical Genetics and Genomics (ACMG)/Association of Molecular Pathology (AMP) codes for variant curation.

Carbamoyl phosphate synthetase I (CPS1) deficiency (CPS1D), is a rare autosomal recessive disorder, characterized by life-threatening hyperammonemia. In this study, we presented the detailed clinical features and genetic analysis of two patients with neonatal-onset CPS1D carrying two compound heterozygous variants of c.1631C > T (p.T544M)/c.1981G > T (p.G661C), and c.2896G > T (p.E966X)/c622-3C > G in CPS1 gene, individually. Out of them, three variants are novel, unreported including a missense (c.1981G > T, p.G661C), a nonsense (c.2896G > T, p.E966X), and a splicing change of c.622-3C > G. We reviewed all available publications regarding CPS1 mutations, and in total 264 different variants have been reported, with majority of 157 (59.5%) missense, followed by 35 (13.2%) small deletions. This study expanded the mutational spectrum of CPS1. Moreover, our cases and review further support the idea that most (≥90%) of the mutations were \"private\" and only ∼10% recurred in unrelated families.

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