Fatty acid hydroxylase-associated neurodegeneration (FAHN/SPG35) is caused by pathogenic variants in FA2H and has been linked to a continuum of specific motor and non-motor neurological symptoms, leading to progressive disability. As an ultra-rare disease, its mutational spectrum has not been fully elucidated. Here, we present the prototypical workup of a novel FA2H variant, including clinical and in silico validation. An 18-year-old male patient presented with a history of childhood-onset progressive cognitive impairment, as well as progressive gait disturbance and lower extremity muscle cramps from the age of 15. Additional symptoms included exotropia, dystonia, and limb ataxia. Trio exome sequencing revealed a novel homozygous c.75C>G (p.Cys25Trp) missense variant in the FA2H gene, which was located in the cytochrome b5 heme-binding domain. Evolutionary conservation, prediction models, and structural protein modeling indicated a pathogenic loss of function. Brain imaging showed characteristic features, thus fulfilling the complete multisystem neurodegenerative phenotype of FAHN/SPG35. In summary, we here present a novel FA2H variant and provide prototypical clinical findings and structural analyses underpinning its pathogenicity.

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as “maybe report” after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

Over 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient’s cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.

Variants of uncertain significance (VUS) are considered one of the most significant impediments to the translation of genetic test results into precise clinical recommendations. The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) classification guidelines established a general framework for the assessment and classification of genetic variants; yet, gene-specific specifications are needed to enable better variant classification to reduce the number of VUS. The process of gene-specific adaptations of the ACMG/AMP codes is led and accompanied by ClinGen and implemented by Variant Curation Expert Panels (VCEP). The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) VCEP recently published its specifications for the BRCA1 (HGNC:1100) and BRCA2 (HGNC:1101) genes. We investigated the differences in reclassification between the ENIGMA specifications and the standard ACMG/AMP classification system in a clinical setting. We reclassified 121 VUS identified in these genes with the latest annotation data using the standard ACMG/AMP classification system and recommendations from the Sequence Variant Interpretation and the ENIGMA specifications. To simplify the reevaluation process, we have created a University of California Santa Cruz Genome Browser track hub that displays the exact data points required for variant classification using the ENIGMA VCEP specifications at the exon and variant level (https://genome.ucsc.edu/s/abenet/BRCA.ENIGMA.hg19). By comparing the codes used and their different weighting in the 2 approaches, we were able to demonstrate the superiority of the application of ENIGMA VCEP specifications, which resulted in a dramatic reduction of VUS (83.5% ENIGMA VCEP vs 20% ACMG/AMP + Sequence Variant Interpretation). For the diagnostic analysis of the BRCA1 and BRCA2 genes, the use of the ENIGMA VCEP specifications gives the best possible result in the clinical translation of genetic variants. The University of California Santa Cruz Genome Browser BRCA1/BRCA2 ENIGMA track set significantly simplified the interpretation process.