Digenic Inheritance of a FOXC2 Mutation and Two PIEZO1 Mutations Underlies Congenital Lymphedema in a Multigeneration Family

Abstract

Background: The lymphatic system is essential for maintaining the balance of interstitial fluid in tissues and for returning protein-rich fluids (lymph) to the bloodstream. Congenital lymphatic defects lead to accumulation of lymph in peripheral tissues and body cavities, termed primary lymphedema. To date, only a limited number of individual genes have been identified in association with primary lymphedema. However, variability of age of onset and severity of lymphatic abnormalities within some families suggests that multiple mutations or genes may be responsible, thus hampering efforts to identify individual associated genes.

Methods: Whole exome sequencing (WES) was performed in 4 members of a large multigeneration family with highly variable lymphedema and followed by Sanger sequencing for identified mutations in 34 additional family members. Genotypes were correlated with clinical and lymphangioscintigraphic phenotypes.

Results: WES uncovered 2 different mechanotransducer PIEZO1 mutations and one FOXC2 transcription factor mutation in various combinations. Sanger sequencing confirmed the presence/absence of the 3 variants in affected and unaffected family members and co-segregation of one or more variants with disease. Genetic profiles did not clearly correlate with the highly variable severity of lymphatic abnormalities.

Conclusions: WES in lymphedema families can uncover unexpected combinations of several lymphedema-associated mutations. These findings provide essential information for genetic counseling and reveal complex gene interactions in lymphatic developmental pathways. These can offer insights into the complex spectrum of clinical and lymphatic lymphedema phenotypes and potential targets for treatment.

Keywords: Digenic inheritance; FOXC2; Familial lymphedema; Genotype-phenotype correlations; Jaundice; Lymphangioscintigraphy; PIEZO1.

Figure 1

Six-generation Family 4 pedigree showing lymphedema affected and unaffected family members and presence of PIEZO1 or FOXC2 mutations.

Figure 2

Lymphangioscintigrams (LAS) in multiple family members with varied digenic FOXC2-PIEZO1 and homozygous/heterozygous PIEZO1 allele recessive genotypes (Roman numerals correspond to position in pedigree, Fig. 1). LAS of affected family members demonstrate a spectrum of mild to severe abnormalities, but with a similar pattern of lymphatic refluxing hyperplasia rather than lymphatic hypoplasia. Repeated findings include lymphatic valvular incompetence with reflux, obstructed lymphatic channels in lower legs, and occasionally reflux into genitalia. LAS of upper extremities show a hyperplastic obstructive phenotype without regional lymph node visualization, suggesting more central lymphatic dysplasia and obstruction, although tracer eventually enters the blood circulation as demonstrated by hepatic uptake. (A) LAS of a normal subject for comparison. Top: Upper extremities and torso after radiotracer injections in the hands demonstrate normal appearance of lymphatic channels and regional lymph nodes draining the arms. Bottom: Lower extremities and pelvis/retroperitoneum/abdomen after injection of the feet demonstrate normal appearance of lymphatic vessels and regional lymph nodes draining the legs, pelvis, retroperitoneum, and abdomen. No tracer reflux is noted. Normal uptake is seen in the liver and kidneys. (B) LAS in family member V-22 (homozygous missense PIEZO1) after injection of the feet shows hyperplastic lymphatic vessels and multiple, tortuous vessels as well as normal uptake in the liver. (C) LAS in V-2 (compound heterozygous missense and nonsense PIEZO1) shows distal lymphatic hyperplasia, dermal tracer reflux, and delayed transport. (D) Early LAS (left) in V-3 (compound heterozygous missense and nonsense PIEZO1) shows similar obstructive pattern in the right leg, with distal hyperplasia and dermal reflux and a more hyperplastic pattern with valvular incompetence and dermal reflux in the left leg. Delayed (~4 hours later) LAS (right) shows gradual clearing of tracer from the legs and extensive scrotal tracer reflux, suggesting deep lymphatic rerouting and increased pelvic nodal activity. (E) Early LAS (left) in V-5 (heterozygous FOXC2 and compound heterozygous missense and nonsense PIEZO1) and delayed image (at ~4 hours; right) show delayed tracer transport in the left leg with lymphatic hyperplasia and dermal reflux in the right leg and progressive filling of popliteal node (signifying deep rerouting related to proximal obstruction), tracer reflux into the scrotum, and abnormally increased activity in lower abdomen. (F) LAS in V-9 (compound heterozygous missense and nonsense PIEZO1) shows lymphatic hyperplasia with extensive dermal reflux in both legs and tracer dispersion in the abdomen and mediastinum, suggesting central lymphatic dysplasia and obstruction; (G) LAS of lower and upper extremities in V-37 (compound heterozygous missense and nonsense PIEZO1) demonstrates lymphatic hyperplasia, particularly prominent in the upper extremities, and valvular incompetence with dermal reflux. Panels C-F reproduced with permission of Lymphology, but with current image reinterpretation and genotypic characterization added. See text and the Table for further details.

Figure 3

Clinical and lymphatic imaging phenotypes of 2 additional severely affected family members. Images A-E are from family member V-4 with whole exome sequencing and Sanger-documented compound heterozygous FOXC2 and PIEZO1 heterozygous recessive missense and nonsense mutations. Images in F-H are from a distant family member with presumptive PIEZO1 heterozygous missense and nonsense recessive (based on parental genotypes and 3 normal sisters), who initially presented with fetal hydrops at another hospital. In V-4, lymphangioscintigram (LAS) (A) compared with (B) shows progression from severe to complete lymphatic obstruction on lower-extremity LAS over a 6-year period, with obliteration of hyperplastic lymphatics and resulting image resembling congenital lymphatic aplasia, but with persistence of hyperplastic obstructed lymphatics in the upper extremities, with prominent activity at valves and lack of nodal uptake still in evidence of the original hyperplastic pattern. Photographs demonstrate progression from (C) severe lymphedema to (D) elephantiasis over a similar time period to the LAS in (A) and (B). Panel E shows magnetic resonance imaging of the chest without contrast, with vestigial thoracic duct (arrow) and multiple dilated and tortuous lymphatic collecting channels bilaterally in the chest documenting central lymphatic obstruction. F-H LAS in extended family member VI-6, who presented with fetal hydrops shortly after birth, demonstrated on LAS (F) after hand and foot injections generalized lymphatic dysplasia without any distinct lymphatic channels, but rather extensive diffusion of tracer into subcutaneous tissues and body cavities. (G) Two years later, patient had marked regression of generalized lymphedema, as demonstrated by minimal periorbital edema and mild lymphedema of distal lower extremities. (H) Consistent with the improved clinical presentation, LAS after single left foot injection showed persistent marked dermal diffusion but new visualization of discrete dilated channels in the upper leg extending into the retroperitoneum. Retrograde reflux into contralateral right pelvic channels is noted (arrow), likely indicating persistent central lymphatic obstruction. See text and the Table for further details.