尽管基因检测日益普及，但仍有15%-30%的OCA患者无法获得明确的分子诊断。这背后存在诸多挑战：一方面，许多患者携带的OCA2基因变异属于临床意义未明变异（Variant of Unknown Significance, VUS），其致病性难以判断；另一方面，越来越多的证据表明，单个基因位点上的多个变异（包括常见的、与正常色素表型相关的GWAS位点变异和罕见的致病性变异）可能以“单倍型”的形式组合在一起，共同影响基因功能，从而导致疾病。然而，传统的诊断方法通常孤立地评估单个变异的致病性，忽略了这种“组合效应”。在OCA2基因位点，GWAS研究已发现多个与皮肤、眼睛颜色、皮肤癌易感性等相关的常见变异，如rs1800404（导致p.Ala355=，影响剪接）和rs12913832（位于HERC2基因内，是OCA2的远端增强子元件，影响表达）。这些常见变异单独存在时不足以致病，但它们与罕见变异“联手”时，是否会“雪上加霜”，共同促成OCA2表型？这正是本研究旨在回答的核心科学问题。
研究记录了28种不同的单倍型，其中一个或多个罕见错义或剪接变异与GWAS变异c.1065G>A (p.Ala355=) [rs1800404]顺式存在。其中最常见的是p.[Ala355=;Val443Ile]单倍型。值得注意的是，位于rs1800404附近（80 bp内）的剪接VUS c.1116+5G>A，当与rs1800404-A形成单倍型时，其对5‘供体剪接位点的影响比单独c.1116+5A更严重。模拟分析显示，绝大多数与c.1065A顺式的变异单倍型会降低外显子10剪接位点的强度。A haplotype on variant impact on exon 10 splice site strength. The strength of 3′SS (A) and 5′SS (B) was quantified with SplicePort in the presence of 477 simulated individual variants (x-axis) and for the same variants in cis with the c.1065G>A mutation. The variants were obtained by mutating each of the 80 positions (with the exception of the c.1065G>A position) flanking the consensus splice site dinucleotide to all possible three alternative alleles. The vertical and horizontal dashed lines correspond to the reference score of the SS (1.5194 for the 3′SS and 1.28537 for the 5′SS in figure A and B, respectively) and allow for easy assessment of variant impact on splice site strength relative to the reference sequence—decreased scores are generally interpreted as resulting in splicing aberrations (e.g., exon skipping, alternative splice sites), whereas increased scores can be interpreted as leading to higher rates of exon 10 retention. The majority of variants are located below the diagonal dashed line representing cases where the c.1065G>A haplotype decreases the strength of the splice site relative to the simulated variants alone. Five or six cases where the variant;c.1065G>A containing haplotype lead to higher scores are located above the diagonal (shown in green). The c.1116+5A variant is highlighted in red. The larger blue dot corresponds to the 3′ or 5′SS score with the reference sequence (x-axis) relative to the c.1065G>A variant (y-axis). (C) Exon 10 variant schematic noting the location of color-coded variants presented in panels A and B. 3′SS and 5′SS consensus dinucleotides are noted in black boxes.">
