Abstract：
BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2.
OBJECTIVE: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis.
METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay.
RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function.
CONCLUSIONS: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.
OBJECTIVE: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis.
METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay.
RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function.
CONCLUSIONS: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.
摘要：
背景：MME（膜金属内肽酶）中的功能丧失变体是隐性Charcot-Marie-Tooth神经病（CMT）的已知原因。一种深内含子变体，MMEc.1188+428A>G(NM_000902.5)，通过使用seqr平台对两个具有轴突CMT隐性遗传的澳大利亚家族进行全基因组测序（WGS）鉴定。在家族1中以纯合状态检测到MMEc.1188+428A>G,并且在家族2中以具有已知致病性MME变体(c.467del;p.Pro156Leufs*14)的复合杂合状态检测到。
目的：我们旨在通过分离和剪接分析确定MMEc.1188428A>G变体的致病性。
方法：使用体外外显子捕获测定法评估深内含子MME变体c.1188+428A>G的剪接影响。
结果：外显子捕获实验证明，MMEc.1188+428A>G变体产生了一个新的剪接供体位点，导致在MME外显子12和13之间包含一个83bp的假外显子。预测将假外显子掺入MME转录本中会导致MME外显子14中的编码移码和提前终止密码子（PTC）（第Ala397ProfsTer47）。这种PTC可能导致MME转录物的无义介导的衰变（NMD），导致致病性功能丧失。
结论：据我们所知，这是引起CMT的致病性深内含子MME变异体的首次报道.这是重要的,因为使用全外显子组测序筛选方法错过了深内含子变体。应该重新评估患有CMT的个体的深层内含子变异，剪接影响被认为与变体的潜在致病性有关。
目的：我们旨在通过分离和剪接分析确定MMEc.1188428A>G变体的致病性。
方法：使用体外外显子捕获测定法评估深内含子MME变体c.1188+428A>G的剪接影响。
结果：外显子捕获实验证明，MMEc.1188+428A>G变体产生了一个新的剪接供体位点，导致在MME外显子12和13之间包含一个83bp的假外显子。预测将假外显子掺入MME转录本中会导致MME外显子14中的编码移码和提前终止密码子（PTC）（第Ala397ProfsTer47）。这种PTC可能导致MME转录物的无义介导的衰变（NMD），导致致病性功能丧失。
结论：据我们所知，这是引起CMT的致病性深内含子MME变异体的首次报道.这是重要的,因为使用全外显子组测序筛选方法错过了深内含子变体。应该重新评估患有CMT的个体的深层内含子变异，剪接影响被认为与变体的潜在致病性有关。
