Genetic Causes of 3 Matthieu J. Schlögel , Pascal Brouillard , Laurence M. Boon , and Miikka Vikkula

Key Points • Panel-based targeted next-generation sequenc- • Careful patient examination for all signs and ing is the most effi cient approach for diagnos- symptoms is important for precise clinical tic screens. diagnosis. • Secondary lymphedema may be infl uenced by • has a high underlying genetic predisposition. genetic heterogeneity. Currently, 20 genes are implicated. • Neonatal , including non-immune Introduction hydrops fetalis, can also be caused by muta- tions in some of these genes. Lymphedema is known since the middle of the • Genetic predisposing factors are unknown for nineteenth century, yet the fi rst genes associated a large fraction of patients. with this condition have been discovered only in • There is large variability in clinical expressiv- the twenty-fi rst century. Since then, more than ity and often incomplete penetrance for all 20 genes have been linked to the development of signs and symptoms. primary lymphedema. Originally discovered using linkage analysis in large families or animal models, the more recent approach using Next- Generation Sequencing (NGS) has allowed to discover genes using smaller families and even sporadic cases. In parallel, detailed in vitro and M. J. Schlögel , M.Sc. • P. Brouillard , Ph.D. in vivo studies on molecular and cellular mecha- Laboratory of Human Molecular Genetics, de Duve Institute, Université catholique nisms involved in lymphangiogenesis have unrav- de Louvain, Brussels , Belgium eled numerous novel functional candidate genes. L. M. Boon , M.D., Ph.D. Primary lymphedema can be present as an Cliniques universitaires Saint-Luc, Center inherited or a sporadic trait. It can be dominant, for Vascular Anomalies , Universite catholique recessive (with consanguinity or not), or linked to de Louvain, Brussels , Belgium the X-chromosome. There is important heteroge- M. Vikkula , M.D., Ph.D. (*) neity in the clinical appearance of lymphedema. Laboratory of Human Molecular Genetics, Primary lymphedema can be the unique sign, de Duve Institute, Université catholique de Louvain, Brussels , Belgium affect different parts of the body (limb(s), arms, hands, head and neck, abdomen, etc.), be unilat- Walloon Excellence in Lifesciences and Biotechnology (WELBIO), Brussels , Belgium eral or bilateral, and appear at different ages at e-mail: [email protected] onset. It can also be part of a complex syndrome,

A.K. Greene et al. (eds.), Lymphedema: Presentation, Diagnosis, and Treatment, 19 DOI 10.1007/978-3-319-14493-1_3, © Springer International Publishing Switzerland 2015

[email protected] 20 M.J. Schlögel et al. some of which are very rare, with only few cases edema when mutated. The penetrance of lymph- reported. edema is though often incomplete, i.e., even Our current knowledge on the environmental though an individual carries a familial mutation, and genetic variability as the cause of primary (s)he does not necessarily have lymphedema. lymphedema is limited. Most of the Mendelian This is also true for the other associated signs and mutations have been identifi ed in a limited num- symptoms listed in Table 3.1 . ber of patients or even only few families used in The cardiovascular system is often affected; the original linkage study, several of the genes varicose veins are not infrequent. Hydrocele can have been identifi ed very recently limiting the be present in at least four of the entities time they have been used in clinical setting for (Table 3.1 ). In the nervous system, symptoms diagnostic screening, the number of patients range from hearing loss to learning diffi culties screened in reports is often very limited, and and macrocephaly. Cutaneous and subcutaneous most screens have been done on gene-by-gene symptoms are frequent, including infection, pap- basis. Moreover, some of the clinical signs may illomas, and cellulitis, many of which are consid- be missed, and thus, the clinical classifi cation is ered secondary. Yet there are subtype-specifi c not necessarily correct. This renders it diffi cult to differences in prevalence (Table 3.1 ). In the mus- have a representative and a comprehensive over- culoskeletal system, syndactyly or camptodac- view of the current state of the art. tyly is observed. Mutations in GATA2 affect the In this chapter, we make an extensive review respiratory system, generating pulmonary alveo- of the medical literature. Clinical data was col- lar proteinosis. Mutations in GATA2, as well as lected for all patients with a proven mutation, IKBKG, also predispose to severe infections. taking into account each mentioned sign and Involvement of the digestive system is relatively symptom. In the presentation, we divide the rare (GJC2 and IKBKG), and renal abnormalities genes (and associated lymphedemas) into two have been reported only in some cases (VEGFR3, groups. The fi rst category includes the genes that FOXC2, and SOX18). Patients with a GATA2 cause lymphedema as the major sign, which is mutation have a susceptibility to hematologic also the reason for medical consultation malignancies. (Table 3.1 ). The second group contains the genes that are related to a usually well-known syn- drome and for which lymphedema is minor sign Genetic Differential Diagnosis: Which (Table 3.2 ). Only a quarter of all cases are Gene to Screen? explained by mutations within the 20 genes. It appears that the historical classifi cation based on The unraveled high genetic heterogeneity in pri- the age of onset, i.e., congenital (at birth or early mary lymphedema has resulted in a high number in life), praecox (teenage years), and tarda (late in of clinical subcategories. Currently 21 geneti- life) is becoming irrelevant, as this does not cor- cally defi ned subgroups, as well as the subgroup relate with the genetic background. Instead, the of undefi ned ones, exist. Moreover, the latter clinical presentation of the signs and symptoms mostly likely involves several genes. Some of the can be helpful to associate the primary lymph- genetically defi ned 21 subgroups have typical edema to the most likely causative gene. signs, the presence of which can help in clinical diagnosis. In addition, familial history (Table 3.3 ) is an important factor in determining candidate Lymphedema as a Major Sign genes for diagnostic screens. The “unique” signs, i.e., those specifi c to one Phenotype of Patients or two genes, are variable. Isolated lower limb lymphedema present at birth (the classic presen- Lymphedema is the major sign for 14 of the tation of Milroy disease) suggests a FLT4/ genes currently known to cause primary lymph- VEGFR3 (FMS-like tyrosine kinase 4/vascular

[email protected] 3 Genetic Causes of Lymphedema 21 ITGA9 (continued) FLT4 FLT4 VEGFC FOXC2 PTPN14 GJC2 GJA1 KIF11 CCBE FAT4 GATA2 SOX18 IKBKG HGF Hydrops (fetalis) Lymphangiectasia Lymphatic anomaly Varicose veins ¶ – Vascular anomaly 36 % Hemangioma – – – Hydrocoele Telangiectasia 7 28 % % Heart 1/6 defect 4/7 ¶ – – ¶ Neurologic – defect – – 35 Microcephaly % – – 49 % – 2/4 Macrocephaly – – – – Ptosis – – – – 1 – Ophthalmologic % problem – Hearing 13 loss – – % – 42 25 – % % – – – – – – – Skin 1/5 infection – – 7 % ¶ Cellulitis – – – – – – 74 % Eczema – 23 – 1/7 – % – – Skin – problem 100 – – % 23 – % – Hair – – – problem – 7/9 – – – – – Nail or toe – – anomaly – – Distichiasis – – – – – – – 19 % – 25 Dental % – 59 anomaly % – – 10 30 % % – 28 – % – – 1/6 – – ¶ – 2/7 ¶ – – – ¶ 3/3 9 – % ¶ – – ¶ – – – 15 – – – 69 19 % – – % % – – – 12 – % – – 17 – 8 % – % – – – – – 91 – – % – – – – – – 1/4 – – – 1/8 – 46 – % – 42 % 33 % 50 – – – % – – 3/3 1/9 – – – 86 – – % – – – – – – – 100 – – % – – – – – 3/5 – – – – 8 – – – % – – ¶ – – 1/2 – – – – – – – – – – – – – – – – – – 7 – % 2/9 – – – – – – – – – – – 7 – % ¶ – – – – – – – – – – – – – – – – – – – – ¶ – – – – – – – – – – – – – 3/5 – – – – – 1/5 78 – % – – – – 5/5 1/2 – – – – – – – – – – – – – – – – – – – Signs and symptoms by mutated gene; lymphedema a cardinal sign Signs and symptoms by mutated gene; lymphedema a cardinal sign Table Table 3.1 System Cardiovascular Lymphedema Sign 81 % 8/8 71 % (139) Nervous 5/7 (10) (120) 100 % Intellectual (7) disability 1/5 51 % 5 % (12) 100 % – Integumentary 9/9 (5) Papilloma 15 (papule) % (75) 3 % (13) 5/6 10 – (9) % 13 % 20 % (110) 3/4 – – (6) 0/4 (70) – – (4) 69 % (12) – 69 % 7/9 – – – ¶ – – – – – – 1/5 – – –

[email protected] 22 M.J. Schlögel et al. ITGA9 studied is in parentheses. ¶ represents symptoms FLT4 FLT4 VEGFC FOXC2 PTPN14 GJC2 GJA1 KIF11 CCBE FAT4 GATA2 SOX18 IKBKG HGF – – – – – – – – – 86 % – 24 % – – Cleft lip and Palate Dysmorphic face Thoracic/vertebral Joint – Syndactyly 4/5 – Choanal atresia – – Bone – 21 % ¶ – – Warts ¶ 7 % ¶ Hematological dysfunction – – – ¶ – – – – – Kidney – – – – – M:F > 2:1 2 % 7/7 – ¶ – – – – – 40 – % – – – 100 – % – – 2 % 9/9 – – – – 4/5 – ¶ – – – – – 27 7 % – % 31 7 % % – 1/5 – – – – – ¶ 2/8 – – – – – – – – ¶ – – – – – 38 – % – – – – 6/9 – – – – – – – – – – – – – – – – – – – – 53 – % – 10 – % – – – – – – – – – – 2/5 – – – – – – – – – (continued) Table 3.1 Table System Musculoskeletal Reduced growth Sign – – Respiratory Immunitary – (139) (10) Pulmonary Infection – (120) Digestive (7) Urogenital – – (12) Gastrointestinal Genitourinary – Malignancy – (5) – The frequency of observation is translated in percentage when the number examined patients was above 10. Number patient (75) – mentioned occasionally – Tumor – (13) – 4 % 69 % – (9) – – 5/9 – (110) – – – (6) 3 % – – (70) – – – – – – (4) – (12) – 8 – % – 1 % – – – – – – – – – – – 8 % – – – 24 % – – 56 – % – – – – – – 98 % – ¶ – – – – 2/2 – 73 % – – – – – – – – –

[email protected] 3 Genetic Causes of Lymphedema 23 RASA1 (314) s studied is in parentheses. ¶ represents symptoms

Hydrops (fetalis) Lymphangiectasia Lymphatic anomaly Vascular anomaly Heart defect – – – Neurological defect 71 Macrocephaly % Ptosis – Ophthalmologic problem – 91 – % Hearing – loss 82 % 10 % – Skin problem Nail 91 or % toe anomaly – Dental – anomaly – – 37 – % – – Fetal macrosomia – 45 23 % Dysmorphic % face 93 % Thoracic/vertebral 31 – % 53 – % 90 Limb – % defect – – – – 34 – % 99 % – – – Hematological 3/8 dysfunction – 50 % – 77 % – – – – 69 – – – % 19 Renal % defect 3/5 ¶ – 59 – % – – – ¶ – 7/8 – ¶ 79 – – % – – 82 % 53 % 7 63 – % % 38 % – – – – 48 – % 0.60 ¶ % – 31 97 % % 82 % 0.5 – 44 % ¶ % ¶ – 0.60 0.60 % % 4/8 – – – – 60 % 4/5 – – – – – – – – – – – ¶ – – – – – – – – – 40 – % – – Signs TSC1 (>100) TSC2 (>100) PTPN11 (31) SOS1 (56) KRAS (26) Monosomy X (>500) Signs and symptoms by mutated gene; lymphedema a non-cardinal sign Signs and symptoms by mutated gene; lymphedema a non-cardinal sign Cardiovascular Lymphedema Nervous 4 % Intellectual disability 4 % Integumentary 49 % Papilloma (papule) 16 % Musculoskeletal 83 % Reduced growth – 30 % 27 % Respiratory ¶ – Immune – Pulmonary 10 % Digestive Urogenital Warts 36 % – 92 – % Malignancy Gatrointestinal Genitourinary The frequency of observation is translated in percentage when the number examined patients was above 10. Number patient anomaly mentioned occasionally – Tumor 11 % – 56 % – ¶ – – – 29 % – – 0.60 % 42 – % – 83 % – – 27 % 42 % 69 % 38 % – 3/6 – ¶ 5/8 – 2/3 – – – – – 22 % – – – – – – – – – – ¶ Table Table 3.2 System

[email protected] 24 M.J. Schlögel et al.

a Patients ivating 139 ivating lymphedema LE gain-of-function, GOF dominant-negative, D-N Genes (proteins) OMIM diseases Inheritances Lymphatic anomalies Types of mutations KIF11 KIF11 (EG5) 152,950 AD, de SOX18 novo IKBKG Lower-limbs LE 607,823 LOF 300,301 AD, AR X-linked LE 75 LE RASA1 608,354 LOF/D-N Hypomorphic AD 6 70 LE LOF 314 loss-of-function, ciency, ciency, LOF autosomal recessive,

AR Diseases and genes associated with primary lymphedema Diseases and genes associated with primary lymphedema autosomal dominant, Number of patients studied Question marks indicate that mutation type and/or inheritance is unclear Table Table 3.3 Diseases a Primary congenital lymphedema/Nonne–Milroy lymphedema Milroy-like (VEGFR3) FLT4 disease 153,100 Lymphedema–distichiasis syndrome (LD) Choanal atresia/lymphedema syndrome AR, de novo AD, Bilateral congenital LE Hereditary lymphedema IC (Meige disease) Inact Oculodentodigital dysplasia/lymphedema syndrome Mental Microcephaly Chorioretinopathy Lymphedema Retardation syndrome (MCLMR) Hennekam lymphangiectasia–lymphedema syndrome Hennekam lymphangiectasia–lymphedema syndrome FOXC2 syndrome) Primary lymphedema with myelodysplasia (Emberger GJA1 GATA2 (CX43) GJC2 (CX47) Hypotrichosis–lymphedema–telangiectasia (renal defect) 613,480 PTPN14 164,200 syndrome (HLTR) CCBE1 Ectodermal dysplasia, anhidrotic, with immunodefi 614,038 153,400 FAT4 osteopetrosis, and lymphedema (OLEDAID) – AD 613,611 Lymphedema–lymphangiectasia VEGFC AD, de novo 235,510 Fetal AD, de novo Tuberous LE sclerosis-1 (TSC1) 235,510 AR Tuberous sclerosis-2 Late-onset (TSC2) LE – AR, de Noonan novo 4-limbs late-onset LE LE syndrome 1 (NS) Missense Noonan AR syndrome 4 (NS) 4-limbs Noonan LE syndrome 3 (NS) LOF Turner syndrome LE Capillary malformation—arteriovenous malformation 12 LOF AD syndrome Weber including Parkes (CM-AVM), 4-limbs LE AD HGF LOF Missense 120 110 Bilateral congenital LE TSC1 LOF LOF LOF 5 – TSC2 13 ITGA9 PTPN11 (SHP2) 191,100 163,950 SOS1 613,254 7 KRAS 10 9 AD? – AD, AD de novo AD, de 610,733 novo Monosomy X 609,942 LE LE LE – AR, de novo AD Nuchal AD edema Hydrops fetalis X-linked GOF Nuchal LOF edema Missense Nuchal LOF? edema LOF Edema GOF 12 31 GOF >100 4 >100 – 56 26 >500

[email protected] 3 Genetic Causes of Lymphedema 25 endothelial growth factor receptor 3) or VEGFC tual disability and eye anomalies, both in 69 % of (vascular endothelial growth factor C) mutation. the patients (MJ Schlögel, Submitted). As for For VEGFR3, the intracellular part is most fre- VEGFR3, failure of initial lymphatic absorption is quently mutated and should be sequenced fi rst observed in lymphoscintigraphy [3 ]. (exons 17–25), whereas any part of the VEGFC Facing a patient with microcephaly, 4-limb coding sequence can be mutated. Functional lymphedema and “unusual face” suggests aplasia of the lymphatic vessels (failure of initial CCBE1 (collagen and -binding EGF lymphatic absorption) in lymphoscintigraphy domain-containing protein 1) and FAT4 (homo- underscores the clinical diagnosis of VEGFR3 log of drosophila tumor suppressor 4) as the mutation. Patients with a mutation in VEGFC mutated gene. This entity is known as the lymph- have reduced uptake with tortuous lymphatic edema–intestinal lymphangiectasia–intellectual tracts and evidence of rerouting [1 –3 ]. disability syndrome or the Hennekam syndrome. When a patient has distichiasis (double rows There is no family history, or history is sugges- of eyelashes, which is not always easy to notice) tive of recessive inheritance. The patient can be and lower limb lymphedema, the likely candidate homozygous or compound heterozygous for the is FOXC2 (forkhead box C2). The lymphedema mutation(s) [7 ]. In one patient with a mutation in is often of late onset, even if some patients with CCBE1, abnormal drainage in the upper and congenital lymphedema or hydrops fetalis have lower limbs, and the thoracic duct, was observed been described [4 ]. The presence of a cleft lip in lymphoscintigraphy [3 ]. and/or palate also guide towards this gene. GATA2 (gata-binding protein 2) and IKBKG Individuals with FOXC2 mutation demonstrate (inhibitor of kappa light polypeptide gene refl ux of lymph within the lower limbs as a result enhancer in B-cells, kinase gamma) are associ- of valve failure within the hyperplastic lymphatic ated with severe immunological problems. vessels [3 ]. Patients with a GATA2 mutation develop warts, Patients with lymphedema on all four extrem- and viral and/or bacterial infections [ 8 ]. ities, whether early or late onset, evoke GJC2 Lymphoscintigraphy reveals hypoplasia of (gap junction protein gamma-2) [5 ]. In lymphos- lymphatics within the affected lower limbs [ 3 ]. cintigraphy, lymphatic tracts appear normal, but Similar features, as well as ectodermal dysplasia, with a signifi cant reduction in absorption by are seen in patients mutated for IKBKG [ 9 ]. It is peripheral lymphatics in all four limbs [3 ]. important to note that GATA2 predisposes to sev- Mutations in another gap junction protein GJA1 eral cancers. This syndrome is known as primary (gap junction protein alpha-1) have been found in lymphedema with myelodysplasia or Emberger patients with lymphedema and, among other syndrome. signs, microdontia; a syndrome known as oculo- There are some additional rare associations. dentodigital dysplasia. The patient had clear In one family, lymphedema was associated lower limb lymphedema and subclinical upper with choanal atresia, and the affected individuals limb lymphedema by lymphoscintigraphy [6 ]. had a partial deletion (exons 7) in PTPN14 Microcephaly can be helpful as a sign for differ- (protein-tyrosine phosphatase nonreceptor-type ential diagnosis. Within the genetically defi ned 14) [10 ]. Another rare syndrome combines groups, it is described, as a major sign, in patients lymphedema with hypotrichosis and telangiecta- with a mutation in KIF11 (kinesin family member sias (HLT = Hypotrichosis–lymphedema–telangi- 11). This phenotype combines microcephaly with or ectasia). It is caused by dominant or recessive without chorioretinopathy, lymphedema, and intel- mutations in SOX18 (SRY-Box 18) [11 ]. A par- lectual disability into a syndrome, abbreviated as ticular stop codon in this gene causes severe MCLMR. Microcephaly is present in 91 % (68/75) glomerulonephritis leading to end-stage renal of the patients and two other major signs, intellec- disease necessitating renal transplantation [12 ].

[email protected] 26 M.J. Schlögel et al.

included in Table 3.2 . With enlarged screens it Lymphedema as a Minor Sign may become evident that the other genetic sub- types can also be associated with lymphedema. In some well-known syndromes, such as tuber- Patients with RAF1 (Raf-1 proto-oncogene, ser- ous sclerosis, Noonan syndrome and Turner syn- ine/threonine kinase) mutation have been drome, lymphedema can be present, although the reported with lymphangiectasia or microkystic diagnosis is made on the basis of other signs and lymphatic malformation. The Costello syndrome, symptoms. In some syndromes, such as in capil- caused by KRAS (Kirsten rat sarcoma viral onco- lary malformation–arteriovenous malformation gene homolog) mutations, shares many features or CM-AVM, presence of lymphedema is only with Noonan syndrome and patients can also rarely reported, and/or it is subclinical, and thus present lymphatic anomalies. Patients with a not systematically looked for. Therefore, preva- PTPN11 (protein tyrosine phosphatase, non- lence fi gures are only weak estimates. These receptor type 11) mutation could have thrombo- genes are presented in Table 3.2 . cytopenia, while mutations in SOS1 (son of sevenless homolog 1) or KRAS seem to com- monly cause macrocephaly. Tuberous Sclerosis 1 and 2

Tuberous sclerosis (TSC) is an autosomal domi- Turner Syndrome nant disease characterized by hamartomas in dif- ferent organ systems (skin, brain, heart, etc.). TSC Turner syndrome occurs in 1/2.500–1/3.000 live- affects between 1/6.000 and 1/10.000 individuals born girls. About 50 % have monosomy X (45,X), [13 ]. Compared to patients with a TSC1 mutation, and 5–10 % have a duplication of the long arm of those with a TSC2 mutation are more likely to one X (46,X,i(Xq)). Most of the rest are mosaic have partial epilepsy, complex partial seizures, for 45,X [ 16]. The main signs are mental retarda- infantile spasms, subependymal giant- cell astro- tion, cardiac disease, renal malformation, short cytomas, and intellectual disability. Dental pits stature, and edema (puffy hands and feet, and are often noted (Table 3.2 ). Primary lymphedema redundant nuchal skin). Most cases of Turner is present in less than 10 % of the cases. syndrome are diagnosed prenatally, by the pres- ence of edema. Karyotype can easily reveal the genetic defect in most cases. Noonan Syndrome

Noonan syndrome (NS) is usually considered as Capillary Malformation– a clinical diagnosis on the basis of the “typical Arteriovenous Malformation face,” including a broad forehead, hypertelorism, Syndrome down-slanting palpebral fi ssures, ptosis, a high- arched palate, and low-set and posteriorly rotated Heterozygous mutations in RASA1 (RAS p21 ears. Cardiac anomalies and cryptorchidism can protein activator 1) cause multiple capillary mal- also be present. Mutations in PTPN11, SOS1, formations (CM) associated with fast-fl ow vas- RAF1, KRAS, NRAS, SHOC2, or CBL cause cular malformations (CM-AVM). There is high this syndrome [14 ]. The overall incidence of intrafamilial phenotypic variability. Almost all lymphatic manifestations among NS is estimated patients with a RASA1 mutation have one or to be ~20 % [ 15]. Lymphedema has only been more capillary malformations (97 %) and 23 % reported in patients with a mutation in PTPN11, have also a fast-fl ow lesion [17 ]. In a few patients SOS1, or KRAS; thus, only these subtypes are with Parkes Weber syndrome and a RASA1

[email protected] 3 Genetic Causes of Lymphedema 27 mutation, primary lymphedema is also present for familial history of the various signs, as they [18 ]. As for the other syndromes in this subclass, could help establish the correct differential diag- the signs of CM-AVM lead to the correct differ- nosis. The usefulness of the novel panel approach ential diagnosis. is particularly appreciated for prenatal genetic testing due to its completeness and rapidity.

Approach for Genetic Screening Genetic Counseling Most diagnostic genetic tests have relied on Sanger sequencing of the exonic parts of a given Diagnostic Genetic Testing candidate gene. When more than one possible candidate gene exists, a sequential approach has The identifi cation of a mutation in one of the classically been used, starting with the gene that known genes allows more precise structure of the most often is mutated in the given clinical sub- follow-up for the patient, especially regarding the type. The high genetic heterogeneity within signs and symptoms that develop with time. For patients with primary lymphedema (so far 20 example, myelodysplasia is not present at birth “candidate” genes) renders this approach time- and necessitates a careful monitoring in patients consuming and labor intense. Only precise clini- with a GATA2 mutation (Table 3.1 ). Genetic cal diagnosis can help target the correct gene. Yet counseling for risk calculation and prenatal the incomplete penetrance of the associated signs genetic testing also become possible via the iden- and symptoms, and the high frequency of de novo tifi cation of the causative genetic mutation. cases for some of the genes may make differential Despite an increased number of genes involved diagnosis impossible. Targeted high- throughput in lymphangiogenesis and/or in the etiology of sequencing now allows to screen several genes at primary lymphedema, a large proportion of a time using panels. This is replacing the sequen- lymphedema patients still remain unexplained tial method. The panel approach allows the clini- after diagnostic genetic testing. Based on the cian to obtain results for multiple genes at once, analysis of more than 400 index patients, muta- which in turn helps in clinical diagnosis, even in tions in the known genes only explain about one the absence of associated symptoms. third of the patients [19 ]. It could be that some of the mutations in these genes go undetected, as they are not in the parts that are classically Prenatal Testing screened (i.e., the exons) or they are not detected by the methods used. These could be intronic or Many signs and symptoms associated with a promoter mutations, or large deletions or inser- mutation in the 20 genes and monosomy X are tions. They would likely not explain the majority detectable only after birth. Thus, differential of the unexplained patients. Thus, additional diagnosis is even more diffi cult in the prenatal genes should exist. period. Fetal edema may appear as nuchal edema, Genetic diagnosis is important to advance our , , chylothorax, pericardial knowledge on primary lymphedema. Precise sub- effusion, cutaneous edema, or hydrops fetalis. classifi cation on the basis of genetic data is These can be caused by mutations in VEGFR3, needed to better defi ne the patient groups for FOXC2, CCBE1, RASA1, PTPN11, and SOS1, genotype–phenotype correlations. Prognosis and Monosomy X. In families at risk for lymph- may also differ between the subgroups. Moreover, edema, detailed morphological ultrasound should targeted therapies are best developed on detailed be carried out paying attention to the signs in comprehension on the underlying pathophysio- Tables 3.1 and 3.2 . It is also important to search logical mechanisms.

[email protected] 28 M.J. Schlögel et al.

Next-Generation Sequencing cation of even the monogenic disease-causing and the Usefulness of a Panel-Based mutation(s) is not without caveats, as it is often Approach diffi cult to make the distinction between an amino acid changing polymorphism and a During the past few years, Next-Generation disease- causing mutation. Sequencing (NGS) has opened a new era for Co-segregation analysis of the identifi ed mutation screens. Whole-exome sequencing changes within the family can give further proof (WES) allows to screen all the coding exons of the for the implication of a given nucleotide change. human genome in one experiment. However, not This can be done using classic Sanger sequenc- all genes are equally well covered. This may be ing. Alternatively, several family members may due to for example inequalities in exome capture be sequenced in parallel using a panel approach. and diffi culty in amplifying areas rich in G and C To predict the impact of a mutation at the pro- nucleotides. Moreover, the cost and ethical issues tein level, different tools based on evolutionary limit the WES approach in clinical settings. conservation, structural constraints, or chemical Another interesting approach is targeted next- qualities of the protein with the changed and generation sequencing. This is based on capture unchanged amino acids have been developed. or amplifi cation of a certain, much more limited, Moreover, databases, such as dbSNP ( http:// number of exons from the human genome. For www.ncbi.nlm.nih.gov/projects/SNP/), example, the 20 known “lymphedema genes” 1000Genomes ( http://browser.1000genomes.org/ cover a total target size of around 117 kb. With a index.html), and GoNL ( http://www.nlgenome. specifi cally designed panel, they can be analyzed nl/search/), collect reference sequence variants in one experiment for a given patient often for a (most of which are polymorphisms) from differ- similar prize as a single gene screen using Sanger ent populations. These can be used to fi lter out sequencing. For the Noonan syndrome, a study known polymorphisms. In contrast, the absence, shows a sixfold reduction in cost using a or a low allele frequency indicates that the variant RASopathy panel with a target area of 30 kb [20 ]. is rare and may have a deleterious impact on the Therefore, it can be used as a primary genetic protein function. Other databases, such as HGMD screen, which does not need a fi nalized, detailed ( www.hgmd.org), regroup the known mutations clinical diagnosis to be effi cient. The value of this in most genes, including small changes (Single technique also lies in its rapidity. Thus, genetic Nucleotide Changes, Multiple Nucleotide results do not only confi rm a clinical diagnosis Changes, insertions, and deletions) and larger but help, in a timely manner, to establish it. structural variations, such as chromosomal deletions, insertions, or amplifi cations. Even with these tools, many identifi ed vari- How to Identify the Causative Variant? ants are reported as having “an unknown signifi - cance.” For these, tests for in vitro and in vivo The Sanger-sequencing-based monogenic functional analysis would need to be developed. screens often reveal one probably pathogenic This is time-consuming and out of scope for rou- variant in one gene. If no such variant is identi- tine diagnostic laboratories. fi ed, screening of a second gene is started. When a “causative mutation” is identifi ed, screens are stopped. This differs fundamentally from the tar- The Lymphedema-Causing Proteins geted NGS approach, which renders data avail- able on all targeted genes at once. Thus, hundreds The genes that harbor mutations causing pri- of variants can be analyzed at the same time, not mary lymphedema and especially the proteins limited to a single gene only. This allows to study they encode can be grouped around the possible interacting variants between the screened VEGFC–VEGFR3 ligand–receptor signaling genes. However, software in diagnostic routine complex and the downstream signaling pathways cannot address this question. Moreover, identifi - via PI3Kinase-AKT and MAPK [21 ].

[email protected] 3 Genetic Causes of Lymphedema 29

VEGFC–VEGFR3 Axis Therapeutic Targets VEGFR3 was the fi rst protein found to be mutated in primary lymphedema patients and the VEGFC– To date, no curative treatment exists for primary VEGFR3 signaling pathway is a major regulator lymphedema. Symptomatic alleviation can be of lymphangiogenesis. VEGFC and PTPN14 achieved by lymphatic drainage, elastic compres- interact directly with VEGFR3. CCBE1 increases sion, and debulking surgery. The risk of infection the capacity of VEGFC to activate VEGFR3 is not negligible and should also be adequately phosphorylation. Downstream of this complex, managed. The numerous associated clinical fea- activation of the transcription factor FOXC2 tures require their specifi c management. ensues. GATA2 also regulates the expression Alternative treatments are being developed. of FOXC2, which plays a major role in the Sorafenib, a tyrosine kinase inhibitor used in development of valves in lymphatic vessels by selected cancers, decreases vascular permeability regulating the expression of connexin CX47/ by suppressing VEGFRs, and it is in a clinical GJC2. Connexin, CX43/GJA1 is enriched on trial for secondary lymphedema [ 25 ]. Another the upstream side of the lymphatic valves. ongoing trial combines autologous Phosphorylation of FOXC2 is also linked to the grafts with adenoviral expression of VEGF-C expression of KIF11 [22 ]. The transcription factor [26 ]. It seems to improve the connectivity of the SOX18 regulates PROX1 (prospero homeobox graft with the . Animal models 1), a main factor for lymphangiogenesis, which are also developed and used to test inventive ther- regulates ITGA9, another valvular protein. So far, apies. Plasmid-based expression of Hepatocyte there is no clear link between VEGFR3 and FAT4, growth factor (HGF) in rat-tail or in mice with but the latter is regulated by the miRNA MIR31, induced upper limb edema inhibits the growth of linked to lymphangiogenesis [23 ]. swelling and lymphangiogenesis [27 ]. As there is high genetic heterogeneity within the causes of primary lymphedema, it would be interesting to Ras/MAPK Axis identify if there is any common pathologic molecular alteration. This would allow a more Another major signaling pathway associated with general, targeted therapy, to be developed. For lymphedema is the RAS-MAPK pathway. example, if the RAS/MAPK pathway would be Mutations in this family of proteins can cause altered in various patients in similar fashion inde- RASopathies. Mutations in each component of pendent of the underlying causative gene, mole- the pathway cause a distinct disease, but all the cules developed to treat cancer could be used for RASopathies share common features, such as RASopathies and primary lymphedema [14 ]. craniofacial dysmorphology and cardiac malfor- mations [14 ]. The pathway is involved in cell cycle, cellular growth, differentiation and senes- Secondary Lymphedema cence. The activation of the pathway can come from a membrane receptor that upon activation Secondary lymphedema is the most common binds adaptors (PTPN11/SHP2, SOS1, and form of lymphedema. It may be caused for exam- RASA1), which increase the proportion of the ple by infection, surgery, radiation, or injury. active form of a RAS protein (KRAS, HRAS). Secondary lymphedema occurs in approximately The activated RAS is able to activate the MAPK 30 % of breast cancer patients who undergo sur- (RAF1) signaling cascade. The MAPK phos- gery or irradiation. Risk factors include the extent phorylates, among others, TSC1 and TSC2, and of surgery and irradiation, disease related factors inhibits their function [24 ]. (stage at diagnosis, pathological nodal status, and

[email protected] 30 M.J. Schlögel et al. number of dissected lymph node), and patient- 3. Connell F, Brice G, Jeffery S, Keeley V, Mortimer P, related factors (age at diagnosis, body mass Mansour S. A new classifi cation system for primary lymphatic dysplasias based on phenotype. Clin Genet. index, and presence of a sedentary lifestyle). A 2010;77(5):438–52. study suggests a link between germline muta- 4. Finegold DN, Kimak MA, Lawrence EC, Levinson tions in CX47/GJC2 and the occurrence of sec- KL, Cherniske EM, Pober BR, et al. Truncating muta- ondary lymphedema [28 ]. Another study tions in FOXC2 cause multiple lymphedema syn- dromes. Hum Mol Genet. 2001;10(11):1185–9. genotyped 155 patients and 387 controls without 5. Ferrell RE, Baty CJ, Kimak MA, Karlsson JM, lymphedema for 17 candidate genes (including Lawrence EC, Franke-Snyder M, et al. GJC2 mis- FOXC2, HGF, VEGFC, and VEGFR3, but not sense mutations cause human lymphedema. Am J GJC2) [29 ]. A signifi cant association was found Hum Genet. 2010;86(6):943–8. 6. Brice G, Ostergaard P, Jeffery S, Gordon K, Mortimer with LCP2 ( cytosolic protein 2), PS, Mansour S. A novel mutation in GJA1 causing NRP2 (neuropilin 2), SYK ( tyrosine oculodentodigital syndrome and primary lymphoe- kinase), VCAM1 (vascular cell adhesion mole- dema in a three generation family. Clin Genet. cule 1), FOXC2, and VEGFC. However, other 2013;84(4):378–81. 7. Alders M, Al-Gazali L, Cordeiro I, Dallapiccola B, studies are needed to confi rm the signifi cance of Garavelli L, Tuysuz B, et al. Hennekam syndrome can be these associations and to identify the nucleotide caused by FAT4 mutations and be allelic to Van Malder changes that are causative for the predisposition. gem syndrome. Hum Genet. 2014;133(9):1161–7. 8. Spinner MA, Sanchez LA, Hsu AP, Shaw PA, Zerbe CS, Calvo KR, et al. GATA2 defi ciency: a protean disorder of hematopoiesis, lymphatics, and immunity. Conclusion Blood. 2014;123(6):809–21. 9. Kawai T, Nishikomori R, Heike T. Diagnosis and Although more than 20 genes have been found to treatment in anhidrotic ectodermal dysplasia with immunodefi ciency. Allergol Int. 2012;61(2):207–17. be mutated in patients with primary lymphedema, 10. Au AC, Hernandez PA, Lieber E, Nadroo AM, Shen they explain less than a third of all cases. YM, Kelley KA, et al. Protein tyrosine phosphatase However, all the 20 genes have never been PTPN14 is a regulator of lymphatic function and cho- exhaustively screened for any patient cohort, and anal development in humans. Am J Hum Genet. 2010;87(3):436–44. the respective prevalences are likely underesti- 11. Irrthum A, Devriendt K, Chitayat D, Matthijs G, Glade mated. Moreover, detailed genotype–phenotype C, Steijlen PM, et al. Mutations in the transcription correlation studies have not been exhaustive, factor gene SOX18 underlie recessive and dominant especially when lymphedema was not the major forms of hypotrichosis-lymphedema-telangiectasia. Am J Hum Genet. 2003;72(6):1470–8. feature of the disease/syndrome. Although it 12. Moalem S, Brouillard P, Kuypers D, Legius E, Harvey remains important to look for all additional signs E, Taylor G, et al. Hypotrichosis-lymphedema- to orient diagnosis, the targeted panel approach, telangiectasia-renal defect associated with a truncat- which allows to obtain results for all known ing mutation in the SOX18 gene. Clin Genet. 2014. Article fi rst published online: 16 APR 2014, genes at ones, will greatly help primary diagnosis DOI: 10.1111/cge.12388. and genotype–phenotype correlation studies. 13. Geffrey AL, Shinnick JE, Staley BA, Boronat S, Thiele EA. Lymphedema in tuberous sclerosis com- plex. Am J Med Genet A. 2014;164A(6):1438–42. 14. Rauen KA. The RASopathies. Annu Rev Genomics References Hum Genet. 2013;14:355–69. 15. Romano AA, Allanson JE, Dahlgren J, Gelb BD, Hall 1. Balboa-Beltran E, Fernandez-Seara MJ, Perez- B, Pierpont ME, et al. Noonan syndrome: clinical fea- Munuzuri A, Lago R, Garcia-Magan C, Couce ML, tures, diagnosis, and management guidelines. et al. A novel stop mutation in the vascular endothelial Pediatrics. 2010;126(4):746–59. growth factor-C gene (VEGFC) results in Milroy-like 16. Sybert VP, McCauley E. Turner’s syndrome. N Engl J disease. J Med Genet. 2014;51(7):475–8. Med. 2004;351(12):1227–38. 2. Gordon K, Spiden SL, Connell FC, Brice G, Cottrell 17. Revencu N, Boon LM, Mendola A, Cordisco MR, Dubois S, Short J, et al. FLT4/VEGFR3 and Milroy disease: J, Clapuyt P, et al. RASA1 mutations and associated phe- novel mutations, a review of published variants and notypes in 68 families with capillary malformation-arterio- database update. Hum Mutat. 2013;34(1):23–31. venous malformation. Hum Mutat. 2013;34(12):1632–41.

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18. Burrows PE, Gonzalez-Garay ML, Rasmussen JC, transduction. Trends Cardiovasc Med. 2014;24(3): Aldrich MB, Guilliod R, Maus EA, et al. Lymphatic 121–7. abnormalities are associated with RASA1 gene muta- 25. Moncrieff M, Shannon K, Hong A, Hersey P, tions in mouse and man. Proc Natl Acad Sci U S A. Thompson J. Dramatic reduction of chronic lymphoe- 2013;110(21):8621–6. dema of the lower limb with sorafenib therapy. 19. Mendola A, Schlogel MJ, Ghalamkarpour A, Irrthum Melanoma Res. 2008;18(2):161–2. A, Nguyen HL, Fastre E, et al. Mutations in the 26. Honkonen KM, Visuri MT, Tervala TV, Halonen PJ, VEGFR3 signaling pathway explain 36% of familial Koivisto M, Lahteenvuo MT, et al. Lymph node trans- lymphedema. Mol Syndromol. 2013;4(6):257–66. fer and perinodal lymphatic growth factor treatment 20. Lepri FR, Scavelli R, Digilio MC, Gnazzo M, Grotta for lymphedema. Ann Surg. 2013;257(5):961–7. S, Dentici ML, et al. Diagnosis of Noonan syndrome 27. Saito Y, Nakagami H, Morishita R, Takami Y, Kikuchi and related disorders using target next generation Y, Hayashi H, et al. Transfection of human hepatocyte sequencing. BMC Med Genet. 2014;15:14. growth factor gene ameliorates secondary lymph- 21. Brouillard P, Boon L, Vikkula M. Genetics of lym- edema via promotion of lymphangiogenesis. phatic anomalies. J Clin Invest. 2014;124(3):898–904. Circulation. 2006;114(11):1177–84. 22. Ivanov KI, Agalarov Y, Valmu L, Samuilova O, Liebl 28. Finegold DN, Baty CJ, Knickelbein KZ, Perschke S, J, Houhou N, et al. Phosphorylation regulates Noon SE, Campbell D, et al. Connexin 47 mutations FOXC2- mediated transcription in lymphatic endothe- increase risk for secondary lymphedema following lial cells. Mol Cell Biol. 2013;33(19):3749–61. breast cancer treatment. Clin Cancer Res. 23. Wu YH, Hu TF, Chen YC, Tsai YN, Tsai YH, Cheng 2012;18(8):2382–90. CC, et al. The manipulation of miRNA-gene regula- 29. Miaskowski C, Dodd M, Paul SM, West C, Hamolsky tory networks by KSHV induces endothelial cell D, Abrams G, et al. Lymphatic and angiogenic candi- motility. Blood. 2011;118(10):2896–905. date genes predict the development of secondary 24. Sevick-Muraca EM, King PD. lymphedema following breast cancer surgery. PLoS abnormalities arising from disorders of Ras signal One. 2013;8(4):e60164.

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