De novo novel splice‐site mutation in FLT4/VEGFR3 is associated with Milroy disease

Abstract

Dear Editor, Milroy disease (MD; Online Mendelian Inheritance in Man #153100), first described by Milroy in 1892, is a congenital developmental disorder caused by dysfunction of the lymphatic system mainly in the extremities. The typical symptom of MD is chronic, asymptomatic lymphedema predominantly in the lower limbs, exhibiting large caliber great saphenous veins with skin manifestations including hyperkeratosis, thickening, papillomatosis, recurrent cellulitis and small dysplastic, upslanting toenails (also called "ski-jump" toenails). MD is usually inherited in an autosomal dominant manner, harboring a mutation in the gene encoding vascular endothelial growth factor receptor 3 (VEGFR3), which is also known as Fms-related receptor tyrosine kinase 4 (FLT4). 1 FLT4/VEGFR3 is essential for lymphatic development. 2 Most studies of FLT4/VEGFR3 in MD revealed missense mutations within the tyrosine kinase domains, which affect lymphatic development. 3,4 Herein, we report a 1-year-old Japanese female infant with MD, who was referred to our clinic with a complaint of edema in her extremities at birth. She was the only child from healthy parents, and no similar symptoms were present in her relatives. She exhibited non-pitting edema in her fingers and the dorsa of her hands, feet and toes with ski-jump toenails (Fig. 1a). Using her blood cells, direct DNA sequencing of the intronexon boundaries flanking exons 17-26 of FLT4/VEGFR3, encompassing two tyrosine kinase domains, where all mutations are located thus far, 4 revealed two mutations in introns 18 (c.2647+1G>T) and 23 (c.3220-35_-2dup34) (Fig. 1b). Interestingly, the latter mutation was shared with the proband's mother, implying a non-pathogenic mutant (Fig. 1c). RT-PCR assay for exons 17-19 and 23-25 revealed a faint but slightly larger band in the patient's product of exons 17-19 (Fig. 1d), suggestive of altered splicing. Sequence analysis revealed that the mutation c.2647+1G>T, which was outside the sequence immediately flanking exon 18, may generate an in-frame mutation with a 36-bp intronic insertion, where a new splice donor site was generated (Fig. 1e and data not shown). As a result, 12 newly added amino acids were predicted as VVGSAGGRG-SLA between those encoded by exon 18 and 19, thereby this mutant VEGFR3 may act in a dominant-negative manner. Reverse transcription polymerase chain reaction (RT-PCR) showed that transcription levels reduced to half of those of her mother and the controls (Fig. 1f). Collectively, these results suggested that the mutation c.2647+1G>T may generate: (i) a mutated FLT4/VEGFR3 with newly added 12 amino acids, showing a dominant-negative effect on the wild-type receptor; or (ii) other altered splice patterns with out-of-frame mutations harboring a premature terminal codon, leading to nonsense-mediated mRNA decay. However, the detailed contribution of this mutation to the development of MD still remains elusive. This is a very rare case of MD with an intronic splice-site mutation in FLT4/VEGFR3, while most previous reports identif...

FLT4 Mutations Are Associated with Segmental Lymphatic Dysfunction and Initial Lymphatic Aplasia in Patients with Milroy Disease

“…Clinical manifestations of MD exhibit considerable variability [2], including within families where affected individuals carry the same mutation. However, de novo cases have been observed in isolated patients; therefore, a family history is not essential for diagnosis [3][4][5]. MD is regarded as a disease caused by lymphatic dysfunction because lymphatic vessels were not visible in most tested patients during lymphoscintigraphic examination; this phenomenon is known as "functional aplasia" because it involves failed isotopic tracer uptake by lymphatic capillaries [6,7].…”