Van Maldergem syndrome (VMLDS/VMS) and Hennekam lymphangiectasia–lymphedema syndrome (HKLLS/HS) are two rare autosomal recessive disorders with highly similar features which were described around three decades ago. Overlapping characteristics of both syndromes include dysmorphic facial features such as epicanthal folds, blepharophimosis, hypertelorism, flat nasal bridge, microstomia besides to microtia, genital involvement, camptodactyly, syndactyly, and developmental delay. However, there are distinctive signs, presented differentially in each syndrome, as lymphangiectasia and lymphedema, which are associated with HKLLS and abnormal findings in brain magnetic resonance imaging (MRI) such as large anterior fontanelles, neonatal feeding, and breath difficulties, which are associated with VMLDS [1, 2, 3, 4]. Tracheal and renal abnormalities are also common among patients with VMLDS [5].
Genetic carrier testing, a method for identifying pathogenic variants in asymptomatic heterozygote parents, emerged in 1970 for hemoglobinopathies. To date, owing to the potency of next-generation sequencing (NGS) methods, it has evolved to expanded carrier screening (ECS), encompassing a broad range of diseases with variable number of genes [11]. Here, we report the usage of whole exome sequencing (WES) for ECS of consanguineous parents with two infants born with multiple congenital anomalies (MCA) and died without exact diagnosis. WES revealed a heterozygous pathogenic variant in the gene
They were two male siblings with a similar set of malformations and their asymptomatic parents were first cousins.
All prenatal screening laboratory tests were normal for both patients and they were born via cesarean section.
Proband 1: He was the first child of a healthy 22-year-old mother and a 26-year-old father, with normal prenatal sonography. After his birth, MCA was observed, including tracheomalacia with tachypnea, leading to tracheostomy in the early weeks after birth. He had facial characteristics of prominent eyes, epicanthal folds, micrognathia, microstomia, and bulbous nose together with short neck, low set ears, microtia with aural atresia and conductive hearing impairment, and clubfoot. No more prenatal or neonatal information is available for this patient.
Proband 2: He was the mother's second delivery at age 24. Several minor abnormalities were found in his fetal sonographies: Nuchal translucency (NT) was 3 mm at gestational age of 12 6/7 weeks. Due to this finding, chromosome analysis of amniotic fluid was performed at gestational age of 15 1/7 weeks. It showed a normal male karyotype 46, XY, which was confirmed by his peripheral blood karyotyping after birth. Prenatal ultrasound at gestational age of 17 3/7 weeks showed breech presentation, normal fetal abdomen, and echogenic bowel. At gestational age of 21 weeks, in addition to echogenic bowel, lateral ventricles were found to be at the upper limit of normal range (9.6 mm). No bowel obstruction or brain abnormality could be found by ultrasound imaging. His birth weight was 2500 g with an Apgar score of 9. Similar to proband 1, after birth he had anomalies of prominent eyes, epicanthal folds, micrognathia, microstomia, and bulbous nose with short neck, low set ears, microtia with aural atresia, and conductive hearing impairment, but limb abnormality was in the form of camptodactyly. In addition, minor cardiac abnormalities were present, including patent foramen ovale (PFO) and mild physiologic tricuspid regurgitation (TR) still in normal ranges. Ultrasound sonography revealed two corticomedullary cysts in his right kidney.
Due to tracheostomy of both of them in the early weeks of life, it is not possible to rule out/in feeding difficulties. The only abnormal finding in laboratory tests of proband 1 was a high level of C-reactive protein (CRP) without evidence of infectious disease. They had respiratory distress and were under ventilator for weeks. Both the patients deceased in the second month of life due to hypoxemia and cardiac arrest.
Carrier screening of parents was carried out using WES. In brief, Twist Human Core Exome Plus Kit (Twist biosciences, San Francisco, CA, USA) was used for exome enrichment and the library was sequenced on Illumina platform (Illumina Inc., San Diego, CA, USA). Mean on-target coverages were 131X and 167X for the mother and father, respectively.
In silico pathogenicity assessments: Allele frequency of the variant was investigated in population databases of 1000genome project (
This study was in conformity with the Helsinki Declaration and ethical approval was not required for this study in accordance with the local/national guidelines. Written informed consent was obtained from the parent of patients for publication of the details of their medical case.
WES data analysis revealed a heterozygous intronic variant, NM_024582.6: c.7018+1G>A, in the gene
The variant was absent from population databases of 1000genome, gnomAD, ExAC, our local database Iranome and, as far as we know, has not been reported before. MutationTaster predicted it as the cause of the disease. In addition, the CADD Phred-score (GRCh37-v1.6 adapted especially for splicing variants) is 33 for this variant, which puts it among the top 0.1% most deleterious substitutions that happen throughout the human genome. We applied splice site prediction tools to analyze the mutant sequence and detect possible cryptic donor sites. All analyses indicated that the CSS is missed and suggested new donor sites;
Comparison of normal CSS, GT, in the intron 6 of
Normal CSS | GATTCAG/ |
+1 | 1.00 | 1–0.83 | −2.074 |
Mutant CSS | GATTCAG/ |
+1 | - | - | - |
Predicted donor sites | CATGGTG/ |
+191 | 0.74 | 0–0.41 | −3.998 |
GACATGG/ |
+683 | 0.99 | 1–0.00 | −2.277 |
CSS, canonical splice site.
Based on criteria from the ACMG guideline, this variant is classified as pathogenic [18, 19]. Applied rules include PVS1 for being a null variant in a gene for which loss of function is the mechanism of disease and PM2 for the variant is not found in the population databases.
In addition to
Table shows variants detected in three genes that parents are heterozygous carriers for them. They share common variants in
c.709A>G p.M237V | Exon 3 | Chr1: 20966418 | Early onset Parkinson disease 6 | 605909 | AR | VUS | |
c.3446-5 dupT | Intron 21 | Chr12: 80542081 | Autosomal recessive deafness 84A | 613391 | AR | Benign | |
Autosomal dominant deafness-73 | 617663 | AD | |||||
c.3622C>T p.R1208C | Exon 3 | Chr17: 18027809 | Autosomal recessive deafness-3 | 600316 | AR | VUS | |
c.5230T>A P.S1744T | Exon 20 | Chr17: 18043849 | VUS |
Variants classification based on ACMG standards and guidelines for the interpretation of sequence variants, 2015.
Exceptionally chromosomal position of PTPRQ gene is presented based on GRCh38 assembly because it has been presented this way in related databases.
Chr. Pos., Chromosomal position; Inh, Inheritance pattern; Loc., Location; VUS, variants with uncertain significance; ACMG, American College of Medical Genetics and Genomics; AD, autosomal dominant; AR, autosomal recessive; CNV, copy number variation; EGF, epidermal growth factor; GRCh38, Genome Reference Consortium Human Build 38; OMIM, Online Mendelian Inheritance in Man; PGD, Preimplantation Genetic Diagnosis; PND, Prenatal diagnosis.
We report two siblings with MCA, born to consanguineous parents, who deceased at the age of two months. During genetic counseling, their parents were explained about a possible familial recessive disorder and informed that in the absence of an accurate clinical diagnosis, a genotype-first diagnosis may be achieved. Therefore, WES was suggested and, due to no samples from their deceased children, it was applied for carrier screening of the parents. Also, they were informed about the advantages and disadvantages of this method. The advantages of WES in ECS, even in cases with no post-mortem samples, have been approved through previous studies. For example, in a recently published report, by this method they could identify the causal variant of a metabolic disorder in three dead siblings. However, as far as we know, in all of them there have been a definite or probable clinical diagnosis. In the current study, no preliminary clinical diagnosis or assumption were given to deceased patients, and from this point of view it could be assumed as the first report of its kind [20, 21, 22].
This approach led to identification of a number of variants shared with the parents, including a novel pathogenic heterozygous variant in the gene
Reverse phenotyping of the patients showed overlapping features of syndromes caused by
Phenotypic comparison of P1 and P2 with HKLLS and VMLDS characteristics previously described in the literature [1, 4, 5, 24]
Microcephaly | + | ++ | No | No |
Large fontanelle | − | +++ | No | No |
Blepharo-nasal malformation | +++ | +++ | Yes | Yes |
Micrognathia and small mouth | +++ | +++ | Yes | Yes |
Irregular dentition | +++ | +++ | NA | NA |
Short stature | +++ | +++ | NA | NA |
Hypertelorism | +++ | +++ | No | No |
Epicanthic folds | +++ | +++ | Yes | Yes |
Camptodactyly | ++ | +++ | No | Yes |
Syndactyly | + | + | No | No |
Clubfoot | + | + | Yes | No |
Microtia | +++ | +++ | Yes | Yes |
Conductive hearing loss | +* | +++ | Yes | Yes |
Cardiac malformation | +* | + | No | Yes |
Infantile hypotonia | +* | +++ | No | No |
Developmental delay | +++ | +++ | NA | NA |
Feeding difficulties | + | +++ | Yes | Yes |
Choanal atresia/stenosis | − | ++ | No | No |
Tracheal anomalies | − | +++ | Yes | Yes |
Periventricular nodular heterotopia | − | ++ | Not performed | |
Corpus callosum anomalies | − | ++ | Not performed | |
Other brain anomalies | +* | + | ||
Renal anomalies | − | +++ | No | Yes |
Genital anomalies | edema | + | No | No |
Lymphedema limb | +++ | +* | No | No |
Primary intestinal lymphangiectasia | +++ | −* | No | No |
Other lymphangiectasia | ++ | − | No | No |
+++, common.
++, around a half of patients.
+, less than half of patients.
+*, a few patients.
−*, only one exception.
HKLLS, Hennekam lymphangiectasia-lymphedema syndrome; VMS: Van Maldergem syndrome; P1, proband 1; P2, proband 2; NA, not applicable.
The Fat cadherin subfamily are highly conserved single-pass transmembrane receptors, which in humans consist of four members FAT1–4. All FAT cadherins have a large extra-cellular domain mainly consisting of cadherin repeats, which are followed by a number of EGF-like and Laminin G-like motifs [25]. Heterophilic interaction of FAT4 with DCHS1, another cadherin, is critical in planar cell polarity (PCP) and Hippo signaling pathways involved in cellular proliferation, orientation, migration, and tissue development [26].
Diagnosis of rare diseases with variable expressivity and overlapping features just based on clinical evaluations is difficult, especially in infantile patients. NGS, as a powerful facility, can unravel genetic defects underlying these group of disorders. Moreover, reverse phenotyping after a genetic finding may extend the clinical assessments and anticipate diagnosis of late onset features [28]. Besides these advantages, NGS has ECS test to hundreds of conditions that makes it increasingly favorable for preconception, preimplantation, or prenatal diagnostic procedures (PND or PGD) [29].
However, next to these advantages, there are routine limitations associated with NGS methods, including the lack of full coverage for all genes or exons, absence of deep intronic regions in WES, and inability to investigate CNVs or chromosomal rearrangements. Moreover, finding variants with uncertain significance (VUS) can bring even more complexity to the situation. Furthermore, there are additional challenges for cases similar to ours: first, the heterozygous variant is identified in healthy adults and cannot be confirmed in a homozygous patient. Second, genotype–phenotype correlation is not straightforward and there is always concern around other possibilities which may be missed. This makes genetic counseling complicated, and any clinical intervention based on these findings must be advised more cautiously [22, 30]
ECS of parents is a powerful test which can reveal the genetic defect in deceased patients suspected to have a rare disease with recessive inheritance pattern. Here, exploiting this method revealed a novel pathogenic splice variant in