Suzanne de Bruijn

57 The impact of modern technologies on molecular diagnostics Other factors that might complicate the interpretation of segregation analysis results are age-related or reduced penetrance, modifiers, carrier females in X-linked disease and multigenic inheritance. Several studies have indicated that modifying variants can have higher allele frequencies than fully penetrant alleles and therefore are not recognized by diagnostic pipelines. 166,167 Despite their high allele frequencies, it has been shown that these variants can still significantly modify Mendelian genotypes. For instance, the variants p.(Ser192Tyr) and p.(Arg402Gln) in TYR have an individual allele frequency of 36.4% and 27.3% in the gnomAD database (non-Finnish Europeans), respectively, while the p.[Ser192Tyr;Arg402Gln] allele has an allele frequency of 1.9%. Despite the relatively high population frequency, the pathogenicity of the p.[Ser192Tyr;Arg402Gln] allele has been suggested when present in homozygous state or in a tri-allelic genotype with a known pathogenic TYR variant in trans . 168,169 Studies suggest that one in six genes implicated in RD is possibly associated with variable penetrance due to variability in expression levels. 170,171 Examples of strong evidence for variants with reduced penetrance implicated in RD or HL have been reported for ABCA4 107,172 , COCH 173 , PRPF31 174 and RIPOR2 . 175 Another complicating factor is uniparental disomy (UPD) where two homologous chromosomes are inherited from the same parent due to errors during meiosis. In 2020, Yauy et al. investigated the presence of UPD in exome sequencing data of 4,912 trios. 176 The authors detected UDPs in 0.05-0.2% of these trios, amongst which was a chromosome 1 UPD ( ABCA4 ) in a Stargardt disease case, suggesting minimal contribution to the genetic diagnostic yield. 176 Thus far, there are four reported Stargardt disease cases showing UPD in chromosome 1. 41,176-178 Moreover, in 2013, Roosing et al. described maternal UPD of chromosome 6, which included a pathogenic TULP1 variant responsible for the cone dystrophy phenotype of the proband. 179 For HL, several cases of UPD have been described as well, affecting chromosome 1 ( USH2A ) 180 , chromosome 13 ( GJB2) 178 and chromosome 18 ( LOXHD1 ). 178,181 Functional evaluation of variants Functional assays can provide an extra line of evidence that can aid in the discrimination between (likely) pathogenic variants, (likely) benign variants or variants with unknown significance. For proteins with a well characterized subcellular localization or function, in vitro approaches can be considered to assess the impact of the variant on protein localization or function. Examples of the latter are assessments of transporter function, enzymatic activity or activity of metabolic pathways. In vivo experiments are ideal for studying the true biological context. However, as it is not always feasible to perform such studies , in vitro research can instead provide valuable information. Biochemical