LYMPHATIC RESEARCH AND BIOLOGY

Volume 00, Number 00, 2018 Original Article

ª Mary Ann Liebert, Inc.
DOI: 10.1089/lrb.2017.0084

Cystic Hygroma: A Preliminary Genetic Study

and a Short Review from the Literature

Giuseppe Noia, MD,1,* Paolo Enrico Maltese, PhD,2,* Giuseppe Zampino, MD,3 Marco D’Errico, MD,4
Vittoria Cammalleri, MD,1 Paolo Convertini, PhD,2 Giuseppe Marceddu, PhD,5
Martina Mueller, BSc,5 Giulia Guerri, PhD,5 and Matteo Bertelli, PhD2,5
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Abstract

Background: The objective of this study is to examine the hypothesis that cystic hygroma (CH) with normal
karyotype can manifest as a Mendelian inherited trait, and that a genetic similitude with hereditary lymphedema
exists. To reach this goal, we investigated the prevalence of genetic variants in angiogenesis and lymphangio-
genesis genes in a cohort of euploid fetuses with CH that almost resolved before delivery. A short review of cases
from literature is also reported.
Methods and Results: Five fetuses were screened using a next-generation sequencing approach by targeting 33
genes known to be associated with vascular and lymphatic malformations. The genetic evaluation revealed two
novel variants in KDR and KRIT1 genes.
Conclusion: A review of the literature to date revealed that an association exists between CH and hereditary
lymphedema and, similar to lymphedema, CH can be inherited in autosomal recessive and autosomal dominant
manner, with the latter most likely associated with a better prognosis. About KDR and KRIT1 genes, no other
similar associations are reported in the literature and caution is needed in their interpretation. In conclusion, we
thought that a genetic test for the outcome of familial CH could be of enormous prognostic value.

Keywords: cystic hygroma, genetic testing, lymphedema, next-generation sequencing, vascular malformations

Introduction The reported rates of abnormal outcome vary and have

been estimated in several smaller studies on 22–134 fetuses
with first-trimester CH.6,8–13 According to these studies, the
C ystic hygroma (CH) is a vascular/lymphatic mal-
formation characterized anatomically by dilated lym-
phatic ducts due to lack of communication between the
rates of abnormal karyotype ranged from 29% to 60%.14 The
rates of congenital anomalies in those with normal karyotype
lymphatic and venous systems. It has an incidence of *1:1000– ranged from 25% to 53%.12 In the absence of karyotype or
6000 births1 and 1:750 miscarriages.2 The malformation de- congenital anomalies, the rates of perinatal death ranged from
velops at the end of the sixth week of gestation3 and is located 3.3% to 11.8%,10,11 and rates of developmental disorders of
predominantly in the neck, accounting for 20%–25% of cervical up to 5.9% have been reported.13
lymphatic tumors.4 Beyond abnormal karyotype, CH is also linked to submi-
The diagnosis of CH is made by ultrasound in the first croscopic chromosomal abnormalities that are typically mis-
trimester of pregnancy.5,6 The syndrome has been associated sed by conventional karyotyping. A comprehensive screening
with chromosomal conditions [trisomy 21, monosomy X (45, tool for genetic syndromes and diseases would therefore be a
X), trisomy 18, and trisomy 13.1,2]6,7 and genetic syndromes. valuable adjunct to current methods of prenatal diagnosis.

1
Hospice Perinatale Centro per le Cure Palliative prenatali Santa Madre Teresa di Calcutta, Policlinico A. Gemelli–Centro Studi per la
Tutela della Madre e del Concepito–Università Cattolica del Sacro Cuore-Roma, Roma, Italy.
2
Magi’s Lab, Rovereto, Italy.
3
Centro Malattie Rare e Difetti Congeniti, Polo Scienza della Salute della Donna e del Bambino, Fondazione Policlinico Universitario A.
Gemelli, Roma, Italy.
4
Divisione di Ostetricia e Ginecologia, Ospedale ‘‘Cristo Re,’’ Roma, Italy.
5
Magi Euregio, Bolzano, Italy.
*These two authors contributed equally to this study.

1
 2 NOIA ET AL.

This may be possible by array comparative genomic hybrid- Custom Capture Enrichment (Illumina) and next-generation
ization technology, which allows submicroscopic genomic sequencing (NGS) on Illumina MiSeq platform (150 bp
alterations to be detected and is becoming standard for the paired-end reads) were performed for genetic analysis of
evaluation of pregnancies with anomalies. all coding exons and flanking exon/intron boundaries of
Interestingly, resolution of nuchal CH in utero as gestation 33 genes known to be associated with vascular and lym-
proceeds has been described in chromosomally normal15 and phatic malformations, as previously described.21,23
abnormal fetuses.16 Souka et al. reported a case of congenital The 33 genes included in the panel were as follows: GJC2
lymphedema of the lower limb manifesting after birth, which (OMIM *608803; NM_020435), KIF11 (OMIM *148760; NM_
showed increased nuchal translucency, but no other structural 004523), FOXC2 (OMIM *602402; NM_005251), CCBE1
abnormalities at 20 weeks of gestation.17 Cytogenetic anal- (OMIM *612753; NM_133459), MET (OMIM *603514;
ysis showed a normal female karyotype. NM_001127500, NM_000245), GATA2 (OMIM *137295;
Cases of familial CH with normal karyotype have been NM_001145662, NM_001145661, NM_032638), FLT4
described and suggest that small inherited genetic variants (OMIM *136352; NM_182925, NM_002020), HGF (OMIM
are involved in the CH phenotype.18–20 *142409; NM_000601, NM_001010931, NM_001010934,
Hereditary lymphedema, also known as primary congeni- NM_001010933, NM_001010932), SOX18 (OMIM *601618;
tal lymphedema (PCL), is a relatively rare condition caused NM_018419), VEGFC (OMIM *601528; NM_005429),
by anatomical or functional defects in the lymphatic system, ABCC6 (OMIM *603234; NM_001171), ACVRL1 (OMIM
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resulting in chronic swelling of body parts. In some families, *601284; NM_000020), AKT1 (OMIM *164730; NM_
the disorder has complete penetrance, while in others, pen- 005163), ANTXR1 (OMIM *606410; NM_032208), CCM2
etrance is incomplete. (OMIM *607929; NM_031443), COL3A1 (OMIM *120180;
PCL as an isolated trait was considered a benign disorder NM_000090), ENG (OMIM *131195; NM_000118), FBN1
with mainly cosmetic impact. Yet, in certain cases, it can cause (OMIM *134797; NM_000138), GLMN (OMIM *601749;
morbidity due to infection and disability due to hindrance of NM_053274), GNAQ (OMIM *600998; NM_002072), KDR
movements. It may also require life-long lymph drainage by (OMIM *191306; NM_002253), KRIT1 (OMIM *604214;
massage or compression therapy. Edema is usually limited to the NM_194456), PDCD10 (OMIM *609118; NM_145860),
upper and lower limbs, but can also affect other districts, such as PIK3CA (OMIM *171834; NM_006218), PTEN (OMIM *0;
the trunk, face, and genitalia. Syndromic forms of varying se- NM_000314), RASA1 (OMIM *139150; NM_002890),
verity also exist. At least 10 genes are associated with the con- SLC2A10 (OMIM *606145; NM_030777), SMAD3 (OMIM
dition and are responsible for about 30% of cases of PCL.21 *603109; NM_005902), SMAD4 (OMIM *600993; NM_
Although CH and PCL are both vascular/lymphatic mal- 005359), TEK (OMIM *600221; NM_000459), TGFB2
formations, very few reports have described a shared genetic (OMIM *190220; NM_003238), TGFBR1 (OMIM *190181;
origin. This is even more the case if we consider isolated CH NM_004612), and TGFBR2 (OMIM *190182; NM_003242).
as a phenotypic trait. A summary of clinical and genetic The bioinformatic analysis was performed using an in-house
findings from case reports in the literature is shown in Table 1. pipeline to align the sequence reads on reference genome,
In this preliminary study, we examined the hypothesis that variant calling, annotation, and variant filtering to remove
CH with normal karyotype can manifest as a Mendelian in- benign single nucleotide polymorphisms (SNPs) (variants with
herited trait. We investigated the prevalence of genetic variants minor allele frequency [MAF] £0.03).
in genes involved in lymphangiogenesis in a cohort of appar- Publicly available databases were used to filter and priori-
ently euploid fetuses with CH. In particular, we considered tize the variants, as 1000 Genomes Project Database (www
genes, which when impaired, are known to be associated with a .internationalgenome.org), Exome Variant Server (EVS) da-
pathological phenotype, specifically primary lymphedema. tabase (http://evs.gs.washington.edu/EVS), Exome Aggrega-
Since lymphatic vessels develop from preexisting vessels tion Consortium (ExAC) database (exac.broadinstitute.org),
during embryogenesis when venous endothelial cells acquire the public database of SNPs (dbSNP, www.ncbi.nlm.nih.gov/
competence to respond to an initial lymphatic-inducing signal,22 SNP), and the Human Gene Mutation Professional Database
impairments in genes involved in the development and main- (HGMD, www.biobase-international.com/product/hgmd), to
tenance of the blood vascular system were also investigated. check for allele frequencies and identify genetic variations
previously reported as pathogenic.
Materials and Methods The potential deleterious effect of missense variants was
determined by using various in silico prediction algorithms
Subjects (SIFT [Sorting Intolerant From Tolerant], PolyPhen-2
For the genetic study, we considered five fetuses with [Polymorphism Phenotyping v2], Mutation Taster, Mutatio-
normal karyotype that during gestation showed evidence of nAssessor, LRT [Likelihood Ratio Test]).
CH, which almost resolved before delivery. Probands were The bioinformatic pipeline, variant prioritization, and san-
followed after birth at least for 1 year (min. 12 months and ger validation of potential pathogenic variants selected have
max. 25 months). Clinical characteristics and follow-up previously been described in detail.21,23
history of cases are reported in Table 2.
Results
Molecular genetics and bioinformatic analyses
Genetic analysis was performed on a group of five unrelated
DNA were extracted from peripheral blood samples with a probands with a history of prenatal CH. We performed NGS
kit based on salting-out (Blood DNA kit E.N.Z.A.; Omega analysis of 33 known vascular and lymphatic malformation-
Bio-Tek). In-solution target enrichment using Nextera Rapid related genes (GJC2, KIF11, FOXC2, CCBE1, MET, GATA2,
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Table 1. Phenotypes and Genotypes of Prenatal Lymphatic Anomalies from Literature

Onset weeks
Reference (PMID) of gestation Prenatal manifestation Postnatal manifestation Diagnosis/Genetics
24
Benard et al. (22021048) 23 Feet edema Feet edema MD/NR
Prefrontal edema
Boudon et al.25 (25896638) 14 Fetal edema of the lower Major edema of the lower VEGFR3 p.(Gly1131Ser)
limbs limbs, hands, and genitalia
Ascites
Boudon et al.25 (25896638) 33 Fetal edema of lower limbs Major feet and legs lymphe- VEGFR3 p.(Pro1137Leu)
(dorsum of the feet and dema
legs) Hydrocele
Daniel-Spiegel et al.26 (16231305) 33 + 4 Polyhydramnios Bilateral feet edema PCL/VEGFR3 p.(Glu1106Lys)
Massive bilateral hydrothorax
Skin and scalp edema
Minimal ascites
Hydrops fetalis
Finegold et al.27 (11371511) UNK Congenital lymphedema CH PCL/FOXC2 p.(His199Profs*264)
Lymphedema

3
Distichiasis
Ptosis
Finegold et al.27 (11371511) UNK Congenital lymphedema Distichiasis PCL/FOXC2 p.(Gly328Valfs*135)
Tetralogy of fallot
Nuchal edema
Franceschini et al.28 (11547763) 19 Legs and feet edema Legs and feet edema NR
Garabedian et al.29 (23074687) 18 Hydrops fetalis Neck edema (postmortem) 1.1Mb deletion in 16q24.1
CH (FOXF1 and FOXC2)
Ghalamkarpour et al.30 (19394045) 12 Generalized subcutaneous Bilateral lower limb lymphe- VEGFR3 p.(Leu893Val)
14 edema dema VEGFR3 p.(Pro1137Leu)
18 Chylous ascites/lower limb Bilateral lymphedema below FOXC2 p.(Gln315*)
edema CH the knees
Left ptosis, distichiasis
Lev-Sagie et al.31 (12528167) 16 Legs edema Legs edema; termination of VEGFR3 (unpublished data)
pregnancy
Makhoul et al.32 (12224079) 15 Feet edema Bilateral partial syndactyly PCL/NR
15 Feet edema Bilateral feet lymphedema PCL/NR
Souka et al.17 (11857608) 13 Legs lymphedema Congenital lymphedema PCL/NR
(continued)
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Table 1. (Continued)
Onset weeks
Reference (PMID) of gestation Prenatal manifestation Postnatal manifestation Diagnosis/Genetics
33
Yang et al. (21584887) UNK Hydropic fetuses with bilat- Intrauterine fetal demise ITGA9 p.(Gly404Ser)/wt
UNK eral chylothorax Intrauterine fetal demise ITGA9 p.(Gly404Ser)/wt
UNK Intrauterine fetal demise ITGA9 p.(Gly404Ser)/wt
UNK Intrauterine fetal demise ITGA9 p.(Gly404Ser)/wt
UNK Intrauterine fetal demise ITGA9 p.(Gly404Ser)/p.(Gly404Ser)
UNK Intrauterine fetal demise PTPN11 p.(Asp61Ala)
UNK Intrauterine fetal demise PTPN11 p.(Tyr62Asp)
UNK Termination of pregnancy VEGFR3 p.(Leu1044Pro)

4
Slee et al.34 (10861674) 23 Polyhydramnios Pneumothorax Suspected YNS (mother affected)/NR
Fetal skin edema Sensorineural deafness
Bilateral pleural effusions Chronic nonproductive cough
Govaert et al.35 (1594340) UNK Nonimmune fetal hydrops Suspected YNS (mother affected)/NR
and recurrent left chy-
lothorax at 4 weeks of age;
mild ankle edema and
marbling of the skin of the
limbs at 1 year of age
CH, cystic hygroma; MD, Milroy disease; NR, not reported; PCL, primary congenital lymphedema; UNK, unknown; YNS, Yellow Nail Syndrome.
 GENETICS OF CYSTIC HYGROMA 5

Table 2. Prenatal and Postnatal Clinical History of Probands

Onset/first
observation Prenatal Resolution Postnatal manifestation Follow-up/last
Case (weeks) manifestation (weeks) (at birth-weeks) observation (months)
P1 16 + 1 Retronuchal, sterna, Postnatal 41 weeks: subcutaneous 22 months: imbibition of the right
and abdominal generalized edema; foot, iris coloboma, angioma
lymphangiomatosis swelling of the right foot on the glabella
P2 22 + 3 Septated CH 32 + 5 41 weeks: healthy 18 months: cystic formation
in the neck
P3 12 Septated CH 16 40 weeks: healthy 12 months: healthy
P4 12 Septated CH 21 + 3 40 + 2 weeks: healthy 25 months: healthy
P5 10 + 2 Septated CH 12 + 4 35 + 4 weeks: healthy 23 months: healthy

FLT4, HGF, SOX18, VEGFC, ABCC6, ACVRL1, AKT1, bilateral thoracic areas, and affecting the medial thighs and
ANTXR1, CCM2, COL3A1, ENG, FBN1, GLMN, GNAQ, right buttock. The right foot showed a tendency to swell.
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KDR, KRIT1, PDCD10, PIK3CA, PTEN, RASA1, SLC2A10, Family medical history included a grandfather with pri-
SMAD3, SMAD4, TEK, TGFB2, TGFBR1, and TGFBR2). mary lymphedema of the lower limbs with onset at 20 years
The average amount of mappable reads per sample was 1.3 M, of age, whereas the father, apparently without lymphedema,
resulting in mean coverage of targeted bases of about had pterygium colli and short neck.
410 · (–21.5) per sample, with 98.4% (–0.11) of bases cov- Additional phenotypic features of the proband were bilat-
ered at least 10 · , and 97.8% (–1.8) at least 25 · . eral coloboma of the iris and angioma affecting the glabella.
A total of seven genetic variants were selected on the basis Molecular analysis showed the heterozygous variant
of the above-mentioned criteria (Table 3). The putative role NM_000118.3: c.572G>A; p.(Gly191Asp) in the gene ENG,
of these variants is discussed below considering phenotype, inherited from the healthy mother.
family history, and pattern of transmission.
Proband 2 (P2)
Proband 1 (P1)
At 22 + 3 weeks, ultrasonographic scan confirmed previ-
Prenatal diagnosis of this female proband indicated ously detected left retrocervical and laterocervical CH. At
generalized systemic lymphangiomatosis with evidence of 27 + 1 weeks, right lateral fold 0.92 mm thick and retronuchal
double laterocervical and dorsal nuchal outline (12 mm) fold 16 mm thick were recorded. At 32 + 5 weeks, the pre-
(Fig. 1). Management was complicated by oligohydramnios viously detected thickening suggesting CH was no longer
in the fifth month. The mother had hypothyroidism, was detectable. Fetal echocardiography was within normal limits.
treated with Eutirox (50 lg/day), and was positive for There were no signs of abnormality at delivery, which oc-
Streptococcus agalactiae B (08/05) treated with incomplete curred at 41 weeks.
intrapartum antibiotic prophylaxis. Delivered at 41 weeks, the At 1 year and 6 months of age, a swelling manifested in
baby showed diffuse edema of the external genitalia and lower the site where the hygroma previously had been detected.
limbs, and subcutaneous formations with parenchymatous/ Ultrasound examination of the neck showed a cystic for-
elastic consistency in the retronuchal, left mandibular, and mation measuring 40 · 17 mm with regular margins and

Table 3. Inheritance, Evaluation of Pathogenicity and Frequencies of Genetic Variants

Nucleotide Amino acid Mutation, 1000
Case Gene Inheritance change change RefSeqa Taster SIFT Polyphen ExACb genomesc EVSd
P1 ENG AD c.572G>A p.(Gly191Asp) rs41322046 DC D PrD 0.0152 0.0032 0.0103
P2 FBN1 AD c.3509G>A p.(Arg1170His) rs137854475 DC T B 0.0012 0.0008 0.0019
MET AD c.2908C>T p.(Arg970Cys) rs34589476 DC D B 0.0029 0.001 0.0035
P3 KDR AD c.3121G>A p.(Val1041Met) NA DC D PrD NA NA NA
P4 KRIT1 AD c.223A>G p.(Thr75Ala) NA DC T B NA NA NA
MET AD c.504G>T p.(Glu168Asp) rs55985569 DC T PoD 0.0037 0.0026 0.0053
P5 CCBE1 AR c.373C>T p.(Arg125Trp) rs147208835 DC D PoD 0.0001 NA 0.00008
a
dbSNP accession number.
b
MAF from the ExAC.
c
MAF from 1000 genomes.
d
MAF from exome variation server.
AD, autosomal dominant; AR, autosomal recessive.
Mutation Taster score system: D, deleterious; DC, disease causing; P, polymorphism; SIFT score system: T, tolerated.
Polyphen-2 score system: B, benign; PoD, possibly damaging; PrD, probably damaging.
dbSNP, database of single nucleotide polymorphisms; EVS, Exome Variant Server; ExAC, Exome Aggregation Consortium; MAF, minor
allele frequency; PolyPhen-2, Polymorphism Phenotyping v2; SIFT, Sorting Intolerant From Tolerant.
 6 NOIA ET AL.

FIG. 1. Ultrasonography at 24 weeks + 3 days showing FIG. 2. Ultrasound picture of nuchal translucency (mea-
evidence of double laterocervical outline, indicated by the sured thickness is of 3.07 mm, as indicated by the arrow).
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arrows, for the presence of cystic hygromas.

noncorpuscular content, and without color Doppler evi- Massive NGS showed the heterozygous variant NM_
dence of vascularization, suspected to be a branchial cyst. 194456.1: c.223A>G; p.(Thr75Ala) in the gene KRIT1 and
The site coincided with a palpable retroauricular cervical NM_000245.3: c.504G>T; p.(Glu168Asp) in the gene MET
swelling. The clinical diagnosis was lymphangioma. Family transmitted by the mother.
medical history was negative for pathologies involving the
lymphatic or arteriovenous systems. Proband 5 (P5)
Genetic testing showed the variant NM_000138.4:
c.3509G>A; p.(Arg1170His) in the gene FBN1 and the Ultrasound evaluation of nuchal transparency at 10 + 2
variant NM_000245.3: c.2908C>T; p.(Arg970Cys) in the weeks showed a bilateral retrocervical and laterocervical
gene MET, transmitted by the father. retronuchal thickness of 3.5 mm, as for septate CH, also with
double outline extending to all of the chest and abdomen
(Fig. 3). This evidence was not found in subsequent exami-
Proband 3 (P3) nations. Family medical history included the father who was
born with retronuchal edema that later regressed.
At 12 weeks, crown rump length (CRL) was 58 mm and a Genetic testing showed a heterozygous variant NM_
sac measuring 18 · 4 mm was detected in the retronuchal re- 133459.3: c.373C>T; p.(Arg125Trp) in the gene CCBE1.
gion. At 13 weeks, CRL was 64 mm and the presence of a
septate retrocervical and laterocervical CH with maximum Conclusion
thickness 2.5 mm was confirmed by ultrasonography. At 16
weeks, CH was no longer detectable and was absent at de- The genetic nature of CH in the absence of an abnormal
livery at 40 weeks. At 1 year of age, the ultrasound picture was karyotype has been postulated several times, because recurrent
normal. No family history of defects was reported. Molecular or hereditary forms have been described with both autosomal
analysis showed the heterozygous variant NM_002253.2: recessive18,20 and autosomal dominant transmission.19,24,25
c.3121G>A; p.(Val1041Met) in the gene KDR. Although the idea that the condition is linked to primary

Proband 4 (P4)
Ultrasound examination at 12 weeks showed increased
nuchal translucency (Fig. 2), as for septate CH with tricuspid
valve regurgitation and atypical ductus venosus blood flow
pattern. At week 18, slight accentuation of the double retro-
nuchal outline, normal ductus venosus flow, and slight ac-
centuation of intestinal echogenicity were found. At 21 + 3
weeks, the general situation had improved, in particular, the
previously reported CH and intestinal hyperechogenicity
were no longer evident; only the nuchal fold was compatible
with the previous diagnosis of hygroma. Fetal echocardio-
gram at 29 + 3 weeks was normal. At 37 + 2 weeks, ultra-
sound imaging did not detect any traces of the previously
detected CH in the retronuchal or lateronuchal areas. When
the baby was delivered at 40 + 2 weeks, there was no evidence FIG. 3. Ultrasonographic evidence of cystic hygroma com-
of hygroma. Family medical history was negative for lym- plicated by fetal hydrops Double outline extending to all of
phatic and venous malformations. the chest and abdomen are shown by arrows.
 GENETICS OF CYSTIC HYGROMA 7

lymphedema (both share the same definition of lymphatic and is presumably associated with the phenotype. KDR is
system dysfunction) has only been expressed by a few authors, expressed in blood and lymphatic vessels, and although it is a
it is an interesting hypothesis.29,30,36 However, no definitive major regulator of physiological and pathological angio-
genotype-phenotype association has yet been established. genesis, some studies suggest that it can also induce lym-
In CH, the developmental abnormalities of the lymphoid phangiogenesis.47,48 In a pedigree with multiple members
system occur at sites where the lymphatic and venous systems manifesting childhood hemangiomas, apparently by autoso-
communicate, thus leading to sequestration of lymphatic mal dominant inheritance (thus hypothetically compatible
tissue.4 These findings suggested that CH could be associated with a variation in the KDR gene), one of the three affected
with genes involved in the organization of both the lymphatic daughters had a CH on her neck.49
and venous systems. We therefore chose to analyze all genes The heterozygous p.(Val1041Met) variation in this gene is
currently known in the literature using an NGS approach. new. We failed to find it in any database of genetic variations
The genetic results are interesting, but caution is needed in and all in silico software predicts it to be pathogenic.
their interpretation, since the variations encountered could be Only the variant p.(Thr75Ala) in the KRIT1 gene was of
merely incidental. interest in proband 4. The variant is novel, not listed in any
There is evidence of hereditary lymphedema in the family questioned database, and two out of three predictors judge it
of proband 1, but no major variations were found in genes to be benign.
associated with this condition. The only variant selected on Heterozygous variations in KRIT1 are associated with
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the basis of the above criteria was p.(Gly191Asp) in the ENG cavernous cerebral malformations (CCMs).50,51 Interest-
gene. Changes in this gene are associated with hereditary ingly, a study investigating the role of the CCM pathway in
hemorrhagic telangiectasia type 1 (OMIM 187300), which the cardiovascular system using mice and zebrafish models
has autosomal dominant transmission. The p.(Gly191Asp) revealed that it regulates endothelial cell-cell association and
variation has been reported in the literature in association mild loss of function in this pathway results in vascular
with gastrointestinal polyps37 and probably arterial pulmo- hemorrhage and lymphatic leakage.52 KRIT1 is also known to
nary hypertension,38 but other studies classified it as non- localize at endothelial cell junctions and regulate their
pathogenic.39–41 All in silico software predicted the variant to function.53 Only one report in the literature seems to indicate
be disease-causing. a possible overlap between lymphatic and CCM phenotypes.
The proband inherited the variant from her healthy mother. The authors described a patient with mixed lymphangioma
Since lymphedema has paternal transmission, since the gene and cavernous hemangioma of the ulnar nerve,54 and al-
shows a total lack of genotype-phenotype association, and though trauma was not excluded as a trigger factor for the
since the variant has a relatively high MAF in the healthy development of lymphangioma, they concluded that alter-
population, we concluded that this variant is nonpathogenic. native mechanisms should be considered. Unfortunately, no
Two variations that could be of a certain interest were genetic testing was performed to elucidate this possibility.
selected for proband 2. Despite conflicting results of in silico Only the variant p.(Arg125Trp) in the CCBE1 gene was
pathogenic prediction software, in the online database, FBN1 found in proband 5. The gene codes for collagen and calcium
p.(Arg1170His) is reported to have a very low MAF and no binding epidermal growth factor (EGF) domain 1 and is as-
homozygote individuals. FBN1 encodes fibrillins and varia- sociated with Hennekam lymphangiectasia-lymphedema syn-
tions in the gene are associated with Marfan syndrome. Fi- drome 1 (OMIM 235510), inherited by autosomal recessive
brillins are a component of lymphatic vessel anchoring transmission. The variant has never been reported to be asso-
filaments involved in opening of interendothelial junctions ciated with a pathogenic phenotype and since it was found in
under conditions of edema to favor interstitial fluid drainage heterozygous state, it is not sufficient to express the phenotype.
and lymph formation.42 However, neither fibrillin knockout It is known that cases of isolated CH, not associated with
mice nor Marfan syndrome patients are described as having aneuploidy or malformations, which resolve by 20 weeks of
lymphatic dysfunction.43 gestation, are likely to result in a normal infant. However, the
The second variant lies in the protooncogene MET. Con- course of a fetus with CH is unpredictable and no long-term
flicting data about the association between MET variations developmental follow-up of infants with resolved hygromas
and the lymphedema phenotype are found in the litera- is available.4 In a previous study, we showed that, among a
ture,21,44 but the interest in CH resides in its interaction with total of 156 cases of isolated CH, 63.6% resulted in a positive
tyrosine-protein phosphatase nonreceptor type 11 encoded by fetal outcome and most cases that demonstrated intrauterine
PTPN11. Variations in this gene are associated with Noonan regression (81.8%) or disappearance (92.6%) also had a
syndrome in which posterior cervical hygroma is a typical positive fetal outcome, as did cases with a normal karyotype
finding. A somewhat high frequency with one homozygote (63.3%). We concluded that, even so chromosomal abnor-
individual is reported for this variant in ExAC database. malities and congenital cardiac pathologies were more fre-
Moreover, there is no agreement about any pathogenic role in quent in CH cases, this does not exclude the possibility of CH
the literature.45,46 All these considerations suggest that nei- spontaneous intrauterine resolution and a good neonatal
ther of these variations are related to the development of CH outcome (Fig. 4).55
in this patient. In our five cases, all pregnancies reached term and the phe-
KDR (also known as FLK1 and VEGFR2) is a gene asso- notype regressed almost completely in all cases before deliv-
ciated with infantile hemangiomas with autosomal dominant ery. Proband 1 had a family history of lymphedema, while the
transmission. Somatic mutations have also been reported. father of proband 5 was suspected to have had CH, since signs
Although no other similar associations are reported in the of excess nuchal skin could still be recognized on his neck.
literature, the KDR p.(Val1041Met) found in proband 3 is, in Two out of three probands (1 and 3) showed complications
our opinion, the most intriguing variant reported in this study, related to lymphatic abnormalities in the first years of infancy.
 8 NOIA ET AL.

Author Disclosure Statement

No competing financial interests exist.

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