Figure 1: PIEZO1 variants in a novel form of hereditary lymphoedema.

(a) Pedigree of the family described in this study. (b) Photographs of II.1 and II.2 at 32 and 7 months of age, respectively. Distinctive facial features include mild infraorbital hypoplasia with subjective hypertelorism, slightly flat facial gestalt with mild periorbital oedema. (c) Photographs of II.1 at 42 months of age. Note abdominal oedema and swelling on the apical surface of the feet. (d) Lateral chest X-ray of II.1 at age 42 months demonstrating pleural effusions. Blunting of the costophrenic angle, a hallmark of pleural effusions, is indicated by an arrow. (e) Alignment of exome sequencing reads (horizontal grey bars) from all individuals to the reference human genome illustrates both the c.3455+1G>A and c.6085G>C mutations. Vertical grey bars next to I.1, I.2, II.1 and II.2 indicate sequencing depth at each base, ∼70 × ; variant bases are highlighted in both the sequencing reads and depth of sequencing bars. The reference sequence and translation are indicated at the bottom of the image. Note that PIEZO1 is encoded by the reverse strand, so variant calls depicted in the sequencing reads are the complement. Predicted protein products for individual family members are depicted below each column of sequencing reads. (f) RT–PCR analysis of patient samples shows defective splicing in those harbouring the c.3455+1G>A mutation. Asterisk indicates shifted product, which is absent in I.2. 5,000 and 50 indicate the internal size standard. (g) Protein alignment of a portion of the C terminus of PIEZO1 illustrating the highly conserved nature of the glycine residue at position 2,029. (h) Sanger sequencing results of RT–PCR products from I.2. (i) Sanger sequencing results of RT–PCR products from I.1. Note the double sequence starting at the exon/intron boundary indicative of retention of intron 24. (j) Deconvolution of dual sequences reveals retention of intron 24 in one allele, with normal splicing from the other allele. (k) Note the software algorithm incorrectly attributes the c.3455+1G>A variant to the exon 25 sequence, so the correct first nucleotide of intron 24 in j is an adenine, while the correct first nucleotide in k is a guanine.