A Milroy Disease Family Caused by FLT4 Gene Mutation of c.2774 T>A with Phenotypes Heterogeneity

Background: Milroy disease is a rare, autosomal dominant disorder. Mutations of FLT4 (Fms Related Tyrosine Kinase 4) gene impaired tyrosine kinase signaling, and further cause symptoms of Milroy disease. In this research, we found a large Chinese MD family with phenotype heterogeneities. And we conducted Next Generation Sequencing analysis to explore possible genetic causative factors might be related to clinical heterogeneities among family members. Methods: Sanger sequencing was conducted on the 17-26 exons of FLT4 (NM_182925.4) gene. Primers were as follows: Froward: 5' CTTCATCAGCGTCGAGTGG 3'; Reverse: 5' ATTATGGGCGGGTTCCTT 3'; Next-generation sequencing was conducted to explore pathogenic mutation might lead to phenotype heterogeneities. Then we conducted Sanger sequencing of the possible related genes. The GIMAP7 gene amplication primers as follows: Forward primer: 5’ ACCACCTGCAAGGAAATCAGCCGCT3’; Reverse primer: 5’GTTAGAGAAATACCTCCTTCCCCTT3’. The amplication system for two genes are as follows: 2×Biotech Power PCR Mix: 10µl; forward primer: 0.8µl (10µM); reverse primer: 0.8µl (10µM); DNA template: 1µl (50ng/µl); ddH20: 13.4µl. The effects of the mutations on the gene functions were evaluated with mutation taster and/or SIFT, PolyPhen. Results: A heterozygous substitution mutation was detected in all patients (FLT4 gene: c.2774 T>A, p.V925E). Meanwhile, a G deletion (c.826delG, p.Val276Phefs*29) of GIMAP7 gene was detected in all patients but two patients with phenotype heterogeneities (I1, and II1). Both the two mutations were predicted to be pathogenic. Conclusions: In this report, we described a large Milroy disease family caused by a missense mutation of the FLT4 suffered from cold and fever-related edema and extended to the thigh, and patient II1 complained the hyperkeratosis and hyperplasia of foot skin from childhood. To explore the possible sources of clinical heterogeneities among family members, we conducted the Next Generation Analysis and found a frame shift mutation (c.826delG, p.Val276Phefs*29) in an immune-related gene : the GIMAP7 gene. Patients carried the frame shift mutation of GIMAP7 gene both have lighter clinical symptoms; So we postulated that the malfunctioned GIMAP7 gene caused by the frame-shift mutation (c.826delG, p.Val276Phefs*29) might inuence immune response, and changed the phenotypes of lymphedema caused by the missense mutation of the FLT4 gene (c.2774 T > A, p.V925E). And further exploration of GIMAP7 gene function should be conducted to testify such assumption.

The propositus with suspected primary lymphedema was self-referred to our research team and was evaluated by lymphological specialists and medical geneticist (Miao Jiang and Wei-tian Han). Then we obtained a complete family history. All participated family members underwent a series of clinical examinations for primary lymphedema. The recruiting criteria of patients are at least one of the following criterions: lower extremity lymphoedema, pitting edema, hyperkeratosis, subcutaneous thickening. We also assessed the morphology of the nail plates. Medical history and physical examination were conducted to exclude the secondary lymphedema: we asked the patient if they have lariasis, cancer, infection, radio-/chemo-therapy and surgery. Genetic counseling offered to family members when the pathogenic mutation was identi ed. Local research ethics committee approved the study protocol and consent forms. All family members enrolled in the study signed two informed consents: one for the genetic test; and second to make the clinical and genetic data available for research purpose.  (Table S10: PCR ampli cation system and conditions of tyrosine kinase coding domains of FLT4 gene). Primer ampli ed exon 20 were as follows: Froward primer: 5' CTTCATCAGCGTCGAGTGG 3'; Reverse Primer: 5' ATTATGGGCGGGTTCCTT 3'; The PCR ampli cation conditions for the FLT4 gene exon 20 ampli cation were as follows: denaturing at 95℃ for 5 min; 35 cycles of denaturing at 95℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 30 s; and nal step for 7 min at 72℃. The ampli ed fragment is 176 bp. The ampli cation system is: 2 × Biotech Power PCR Mix: 10 µl; forward primer: 0.8 µl (10 µM); reverse primer: 0.8 µl (10 µM); DNA template: 1 µl (50 ng/µl); ddH20: 12.4 µl. The ampli cation system is 25 µl. The novel mutation was also ruled out as polymorphism by digestion with the restriction enzyme Hph I and the ampli cation primers are as follows: 5' AACCTCCTCGGGGCGTGCACCAAGC 3', 5' GCGCAGGGGCTGAAGGCGTCCCG 3'; The ampli cation system is: 2 × Biotech Power PCR Mix: 10 µl; forward primer: 0.8 µl (10 µM); reverse primer: 0.8 µl (10 µM); DNA template: 1 µl (50 ng/µl); ddH20: 2.2.2 PCR ampli cation and Sanger sequencing of the possible pathogenic genes If any variant was included, we veri ed it within the family members. We conducted PCR ampli cation and Sanger sequencing in four patients (I1, II1, III3 and III4) and the healthy control (II7). PCR ampli cation and Sanger sequencing analysis based on the variant's information. The PCR ampli cation systems are listed in supplementary materials (Table S8: PCR ampli cation systems and conditions of possible pathogenic variants). The Sanger sequencing was conducted by Sangon Biotech, Shanghai, China.

Mutation analysis
A search performed for human FLT4 and GIMAP7 protein homologs using BLAST on the NCBI web site (http:// www.ncbi.nlm.nih.gov). The identi ed proteins were aligned using ClustalW [13], and a phylogenetic tree was reconstructed with MEGA4 with neighbor-joining method [14]. The effect of the amino acid substitution on the protein function was evaluated with mutation taster (www.mutationtaster.com) and/or SIFT [15,16], PolyPhen.

PPI network generation
First, the PPI networks of FLT4 and GIMAP7 gene were generated using string database (https://string-db.org/cgi/input.pl). Then we construct functional annotation maps to understand which protein was involved in the FLT4 and GIMAP7 gene functions. The interaction network were obtained from the STRING database version 10.5.

Description of the MD pedigree
The family history presents 7 affected patients (age ranged from 29-75 years old) of four generations ( Fig. 1 Pedigree of the family). Table 1 [4]. No asymptomatic carrier, late onset or non-penetrance patient found within the family.   (I1, II1, II2, II4, II6, III3 and III4) but not in  unaffected family members (II3, II5, II7 II8, III1, III2, III5 and IV1). The novel mutation was also ruled out as polymorphism by Hph I digestion, the PCR ampli cation fragement is 262 bp. The restriction endonuclease cleavage site is 5'GGTGA(N) 8 3'. If the mutation site presented, the fragment is cleaved and turn out to be two fragments: 198 bp and 64 bp, and analyzed by 8% polyacrylamide gel electrophoresis and silver staining. Using DL2000 as the marker (Takara). The polypropylene gel electrophoresis voltage is 400 Voltage and the electrophoresis time is 4 hours. (Fig.S1: Hph I restriction enzyme digestion results of FLT4 gene), and no existence in 100 healthy controls showed a normal sequencing pattern. Using the SIFT program the normalized probability of the substitution of V to E was calculated to be 0.00, which was less than the threshold of 0.05. Thus, this mutation was predicted to be deleterious. The PolyPhen program predicted that this mutation was likely to be damaging with a score of 1.00. Meanwhile, the mutation taster website (http://www.mutationtaster.org/) also showed the substitution V925E is disease causing. Twelve proteins in the FLT4 subfamily were found by a BLAST search in NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) formed a cluster in the phylogenetic tree ( Fig.S2: Polygen Tree of FLT4 gene). FLT4 protein sequence alignment revealed the invariant valine in the tyrosine kinase domain and the wild type is conserved in a wide range of organisms, ranging from humans to Danio_rerio   II3, II5, II7 II8, III1, III2, III5  and IV1). Also, no existence in 100 healthy controls showed a normal sequencing pattern. Mutation taster website (http://www.mutationtaster.org/) showed the nucleotide c.826G deletion (p.Val276Phefs*29) is disease causing. Seven proteins of the GIMAP7 subfamily were found by a BLAST search that formed a cluster in the phylogenetic tree ( Fig.S5: Polygenic tree of GIMAP7 gene). GIMAP7 proetin sequence alignment revealed the presence of nucleotide G is conserved in a wide range of organisms, especially within the Primates, ranging from humans to macaca nemestrna ( Fig.S6: conserved motif of GIMAP7 gene).

Discussion:
Primary congenital lymphedema (PCL), also called Milroy disease (OMIM:#153100, MD) is caused by developmental lymphatic vascular anomalies, with an estimated prevalence of 1 in 160,000 individuals [17]. We reviewed the reported phenotypes of the Milroy disease, (Table S9. Review of clinical phenotypes of Milroy disease) that MD patients usually exhibit lymphedema at birth with swelling of the lower-limb, and most are bilateral [2]. They often have brawny texture and hyperkeratosis of foot skin; (Table S9: Review of clinical phenotypes of Milroy disease). The other phenotypes associated with MD including: Hydrocoele of male (37%), "ski jump" toenails (14%) and bilateral pleural effusion [2] (Table S9: Review of clinical phenotypes of Milroy disease). Although below knees lymphedema is the most common phenotypes of MD, edema of some patients extends to thighs [2,18]. Meanwhile, hydrocoele is more common in male patients [2,3,[19][20][21][22].
The FLT4 (FMS-LIKE TYROSINE KINASE 4; FLT4; NM_182925.4) gene mutations cause kinase-inactivation and Milroy disease [6,7,[22][23][24][25][26][27]. to date, all mutations located in either of two intracellular kinase domains [6,7]. In this research, we sequenced the tyrosine kinase coding domains of FLT4 gene in a large hereditary congenital lymphedema family which presents Milroy disease phenotypes, and found a missence mutation of c.T2774A which leads to the Valine to Glutamic acid substitution exchange (Fig. 4 Sanger sequencing result of the FLT4 gene c.2774 T-A mutation. p.V925E). This missence mutation causes the major phenotypes of Milroy disease in our pedigree members.
Interestingly, two patients suffered for speci c clinical phenotypes compared with other patients within the family: patients I1 complaint that edema of his bilegs aggravated and extend to the roots of the thighs when he caught cold and fever from childhood; Meanwhile, patient II1 complained that: as for childhood, edema continued to the lower extremities beneath the knees, and lymph uid caused thinning and deforming of skin tissue, combined with proliferation of adipose and connective tissue, lead to swelling and bulky of feet. The lymphedema phenotype is not visible now due to plastic surgery, but the heavier lower extremities edema and "elephant-like" skin hyperkeratosis appearance of her legs is obvious (Fig. 2 − 1,2: bilateral lower limbs lymphedema of patient II1) [4,28,29]. On the contrary, the phenotypes of the other patients (except I1 and II1) are not like these two patients: lighter below knees lymphedema could be found in most of the patients within our family (Fig. 2-3: bilateral lower limbs lymphedema of patient II6; Fig. 2-4: bilateral lower limbs lymphedema of patient III3), some of the patients' lymphedema is hard to see (II2, II4 and III4), neither the brawny texture of the skin; No hydrocoele has found within the male members.
As for now, reporters believed that the major pathologic changes of Milroy disease are aplastic, hypoplastic and dysfunctional cutaneous lymphatic vessels [6,12], which fail to transport the uid into the venous circulation, result in a lymphatic uid stasis and swelling of the extremities [30][31][32][33] That is, lymphatic vessels malformation triggers the increase of interstitial protein rich uid, results in insu cient lymphatic drainage and transport [34]. As a result, large amount of protein-rich uid accumulates in tissue interstitial spaces, makes skin, subcutaneous tissue, brous tissues hyperplasia, and oppress of lymphatics more di cult to re ux for lymphatic; the edematous uid and adipose tissue accumulate subcutaneously, and followed by in ammatory response developing and forming a vicious cycle that aggravating the formation of edema [19,35]. Meanwhile, slowed down of lymphatic ow incites lipogenesis and fat deposition and leads to increased brocyte activation and connective tissue overgrowth [36][37][38]. Firmer subcutaneous tissue as brosis, hypertrophy of adipose tissue presents, these pathologic changes manifest initially as swelling of the affected limb or region, described as soft and pitting, but later progress to a more indurated state. The skin thickened, hardened, getting rough and bulky, and forming "elephant skin" swelling over time.
In our pedigree, we found two elder patients suffered for fever-related edema (I1) and heavier lower extremities edema and "elephant-like" skin hyperkeratosis appearance (II1), but phenotypes of other patients are not like them. The Heterogeneities of symptoms drove us to conduct NGS analysis to explore possible variants might be related to the phenotypes. Then we found a G deletion (c.826delG, p.Val276Phefs*29) of GIMAP7 gene in all patients except two (I1 and II1), neither in all the healthy controls. To date, there have been very few reports on the gene function of GIMAP7 gene. GIMAP7 gene belongs to the GTPase family, which is a family of immuno-related proteins (GIMAPs, GTPase of immunity-associated proteins), is widely expressed in the immune system and is differentially regulated during early human Th-cell differentiation [39]. The GIMAPs have been closely linked with cell survival of lymphoid cells [40]. Regulation of cell viability of death is likely to be regulated through GIMAP family members in immune cells. And the expression of GIMAP7 gene in normal tissues is higher than that in tumor tissues and may play a regulatory role in the immune response regulation in coordination with other molecules [41]. We speculated that the G deletion (c.826delG, p.Val276Phefs*29) of GIMAP7 gene caused the malfunction and its coordination with other molecules, and nally jeopardized immune response of the body, then the patients in this MD family both showed different symptoms compared to the patients I1 and II1: eg. The immune related-such as cold and fever related edema, and serious "elephant skin appearance" of foot skin even the hyperplasia of foot tissue. On the contrary, in Milroy disease patients--just like the patients I1 and II1 and other reported MD patients--protein-rich uid accumulates in tissue interstitial spaces and followed by in ammatory response developing-forming a vicious cycle that aggravate the formation of edema and hyperplasia, leads to "hyperkeratosis appearance" of foot skin [19,35] and below knees edema. According to the PPI network, the FLT4 and GIMAP7 protein is not directly related, and several proteins were involved in the protein-protein interaction network ( Fig. 6 Protien-Protein interaction network of GIMAP7 and FLT4 gene). And some of the involved gene function is immune-related. Eg. the LILRB2 gene. So we speculated that immune associated gene malfunction and protein interactions might be the main reason for the phenotype heterogeneities. Based on our ndings, our genetics and research team suggested that: Further research on GIMAP7 gene function should be conducted to testify our hypothesis. Two mutations occurred within one family and orchestrated together to present phenotype differences is a new perspective to explore heterogeneities, and need further research and attention. GIMAP7 gene mutation can be used as a potential molecular target for future exploration.

Conclusion:
In this report, we described a large family with the manifestations of Milroy disease which caused by a missense mutation of FLT4 gene (c.2774 T > A, p.V925E). All the patients within the family presented with lighter phenotypes except two (I1 and II1): Patient I1 suffered from cold and fever-related edema and extended to the thigh, and patient II1 complained the hyperkeratosis and hyperplasia of foot skin from childhood. To explore the possible sources of clinical heterogeneities among family members, we conducted the Next Generation Analysis and found a frame shift mutation (c.826delG, p.Val276Phefs*29) in an immune-related gene : the GIMAP7 gene. Patients carried the frame shift mutation of GIMAP7 gene both have lighter clinical symptoms; So we postulated that the malfunctioned GIMAP7 gene caused by the frame-shift mutation (c.826delG, p.Val276Phefs*29) might in uence immune response, and changed the phenotypes of lymphedema caused by the missense mutation of the FLT4 gene (c.2774 T > A, p.V925E). And further exploration of GIMAP7 gene function should be conducted to testify such assumption.