Clinical features and diagnosis
The patient was admitted with characteristics indicating certain type of hereditary lymphedema. All the parameters of general examination including the vitals were normal. Examination of lower limbs revealed bilateral edema of legs and feet with dryness and hyperpigmentation of the overlying skin. Examination of the eye revealed partial ptosis on the left side, extra partial set of eyelashes on both eyes and left corneal opacity with reduced visual acuity on left eye. Slit lamp examination of both eyes showed bilateral distichiasis, epithelial and stromal opacity in the center of cornea and superficial corneal vascularization on the left eye. Results of other systemic examinations were normal. Isotope Lymphoscintigraphy of both lower limbs revealed grade-1 and grade-2 lymphoedema on the left and right side respectively. Findings of serum creatinine, ALT, Prothrombin time, urine R/M/E, ECG, Echocardiogram and CXR were normal. ICT for filaria was also negative. Duplex study of both lower limbs found no evidence of deep vein thrombosis and no evidence of arterial insufficiency.
Based on these clinical features and the patient statement of his father and younger brother having similar illness, he was diagnosed with a rare hereditary disease, Lymphedema distichiasis syndrome.
After necessary documentation, blood sample from the patient was collected to test for the presence of FOXC2 mutations.
PCR results of FOXC2 gene
PCR amplification of the 5 primer sets showed specific bands only for pair number 1 and 5 (Figure: 3a). Regions covered by primer pair one and five were sequenced (Figure:4).
To reveal the sequence between, several modifications were tried. Interestingly, gel electrophoresis indicated amplification from primer combination of F1 and R5 with 5% DMSO (Figure:3b). Annealing and extension temperatures were 600C and 720C respectively for this reaction. Later, this product was used as template to perform nested PCR with primer combination of F2 and R4. In this nested PCR reaction mix, annealing temperature was 600C and 3% DMSO was added considering presence of DMSO in the product to be used as template. Nested PCR with primer combination of F2 and R4 resulted in better amplification but nonspecific products were present (Figure: 3c).
The band of desired size of 1120 bp was isolated using Gel extraction after electrophoresis. This 1120 bp DNA was sequenced using Sanger method from both primers. PCR products of primer pair F1+R1, F2+R4 and pair F5+R5 overlap each other and cover the whole coding region of FOXC2 gene. So, these three products were sequenced to get the complete sequence of the gene (Figure:4).
Sequence analysis of FOXC2 gene: Primer pairs of PCR products that provided good quality sequence are F1+R1, F5+R5 and F2+R4. When blast search against the NCBI nucleotide database were performed, no mutation was found in the sequence derived from pair F1+R1 and F5+R5. Product of primer combination F2+R4 was sequenced with both F2 and R4 primer to get the sequence of the whole segment. Sequence from R4 was reverse complemented and after trimming they were assembled in full 1120 bp sequence. When the assembled sequence was blast searched, a 8 bp deletion (ACGCCGCC) was identified (Figure:5a). The chromatogram also indicates that the patient is homozygous for this deletion (Figure:5b). This deletion is identified to be between base no 6423 and 6431 of RefSeqGene on chromosome 16, (which refers to mRNA position between 914 and 921). This mutation had been reported before for the same disease. ClinVar accession code for this deletion is RCV000007679.3. Although most other mutations of this gene are unique among different families, as of October, 2021 only this 8 bp deletion has been reported four times in four independent studies for families from different geographical region (4,11–13). These changes resulted in the production of a premature stop codon that terminated the predicted protein earlier than the wild-type and produced novel C-termini. Codons after 304 are disrupted for this mutation and new 154 amino acids are added.