Congenital heart anomalies (CHAs) are the most common birth anomalies, and syndromic CHAs represent approximately 25.0% of cases [1,2]. The introduction of molecular karyotyping (molecular chromosome microarrays) and later, next generation sequencing (NGS), in the diagnosis of syndromic CHAs, have enabled the identification of numerous microdeletion and microduplication syndromes as well as single gene disorders as a cause of congenital cardiovascular malformations accompanied by additional features [2]. Representative of the latter is the Kabuki syndrome (KS) with an estimated prevalence of 1/86,000 to 1/32,000 newborns [3]. Kabuki syndrome is a multiple anomaly syndrome characterized by five main features: postnatal growth retardation, dysmorphic facial features, skeletal anomalies, dermatoglyphic abnormalities, and intellectual disabilities (ID) [4]. According to the international consensus diagnostic criteria for KS, a diagnosis of KS can be achieved in probands at any age if the infantile hypotonia, developmental delay (DD) and/or ID was observed, and one or both criteria: molecularly confirmed pathogenic or likely pathogenic variant in causative gene and/or presentation of at least three typical dysmorphic features characteristics for KS at some point in life [4]. The prevalence of CHAs in KS ranges from 28.0 to 80.0% of patients [5].
In approximately 75.0% of patients, the genetic causes of KS are pathogenic or likely pathogenic mutations found in the
In newborns, a clinical diagnosis of KS is often challenging due to phenotype evolving over time. Namely, facial features are most evident in children between the ages of 3 and 12 years [4]. Newborns usually display milder facial features, therefore, the diagnosis is based more on congenital malformations [10]. Moreover, some of the observed features in newborns overlap with features in disorders such as the CHARGE syndrome, 22q11 deletion syndrome, IRF6-related disorders,
Here, we report of a case of a 2.5-year old boy with CHA observed dining pregnancy. Cytogenetic and molecular testing was performed prenatally to exclude aneuploidies and chromosomal structural variations, however, no discrepancies were noted. After birth, the proband was referred for NGS testing, primarily due to the observed CHA. In the first moths, additional features became more prominent in our proband, therefore, an assumption for a single gene genetic disorder was made. After a genotypephenotype driven analysis, a
During pregnancy, CHA was suspected in the fetus. To exclude aneuploidies or microdeletions or microduplication syndromes, the genetic analysis included classic karyotyping, an in-house developed quantitative fluorescent polymerase chain reaction (QF-PCR) method with 20 micro-satellite markers located on chromosomes 13, 18, 21, X and Y, molecular karyotyping with 8 × 60 K BlueGnome CytoChip Oligo (BlueGnome Ltd., Cambridge, Cambridgeshire, UK) and multiplex ligation-dependent probe amplification (MLPA) with the SALSA MLPA P036 probemix (MRC-Holland, Amsterdam, The Netherlands) was used. All the performed prenatal genetic tests were negative. Generally, the mother reported no problems during the pregnancy. The onset of labor was spontaneous and without any complications. The baby’s birth weight was 3010 g (50th-90th centile for gestational age), birth length 49 cm (50th-90th centile), head circumference 31.5 cm (10th centile) and Apgar score was 8/9. After birth, perimembranous ventricular septal defect (VSD) and pala-toschisis were diagnosed and some dysmorphic features were noted. The male newborn had a depressed nasal bridge, arched eyebrows with long eyelashes, epicanthus, long palpebral fissures, and somewhat protruding earlobes. The skull had a biparietal narrowing and the occipital part was flat and high. In addition, general muscular hypotonia was present with very scarce spontaneous movements.
The hemodynamically important VSD was surgically treated after birth. Osteomyelitis of the sternum was identified as surgical complication, therefore, prolonged treatment with antibiotics was administered. Retention of the right testis was persistently present, bilateral sensorineural deafness as well as anomalies of spinal vertebrae were also diagnosed. Ultrasound examination revealed renal pelvis dilation on the left and a bilateral vesicoureteral reflux. Despite intensive neurophysiotherapy, the muscular hypotonia persisted and developmental milestones were delayed. In addition, physiological weight gain was constantly impaired. However, no seizures were noted and magnetic resonance imaging (MRI) showed no structural anomalies of the brain.
At 8 months, serious acute pyelonephritis with extended-spectrum β-lactamases (ESBL) producing
At the time we reached our diagnosis, the proband was 12 months old. He weighed 6.9 kg (below the 3rd centile), his height was 68 cm (below the 3rd centile), and his head circumference was 42 cm (below the 3rd centile). Dysmorphic features were more prominent, muscular hypotonia was still present and a significant DD became evident. He was able to roll over from his front to his back and
Variants obtained after NGS sequencing were analyzed and interpreted according to the American College of Medical Genetics and Genomics (ACMG) guidelines [12]. First, common variants with an allele frequency of >1.0% according to public databases [Genome Aggregation Database (gnomAD), the 1000 Genomes Project (1KGP) and the Exome Variant Server (EVS)] were excluded from the analysis. The remaining variants were then analyzed, predominantly focusing on variants in genes involved in VSD (HP:0001629), microcephaly (HP: 0000252), and muscular hypotonia (HP: 0001252), features described as predominant in the proband. With the aforementioned protocol, we were able to determine the previously unreported frameshift deletion NM_003482.3: c.11093delG in the
After reaching the diagnosis, the proband was reevaluated according to the international consensus diagnostic criteria for KS at age 10 months and 2.5 years [4]. Figure 2 presents mild facial feature of KS observed in our proband during his follow-up at age 2.5 years.
Table 1 represents the most common phenotypic features observed in our proband compared to individuals with reported heterozygous pathogenic variant in the
Comparison of features observed in our proband with the most commonly observed features in individuals with heterozygous pathogenic mutation in the
Phenotypic Features in Patients with | Features Present in Our Proband |
---|---|
Intellectual disability (IQ <70) | not tested |
Fetal fingertip pads | no |
Congenital heart defect | yes |
Long palpebral fissures | yes |
Large, prominent or cupped ears | yes |
Hypotonia | yes |
Eversion of the lower eyelid | no |
Arched or broad eyhrows | yes |
Cleft palate | yes |
Brachydactyly | yes |
Short columella with depressed nasal tip | yes |
Short stature | yes |
Microcephaly | yes |
Oligodontia and/or abnormal incisors | no |
Feeding difficulties | yes |
Developmental delay | yes |
Latent eyebrows, sparse or notched | yes |
Hearing loss | yes |
Non traumatic joint dislocation | no |
Hypogammaglobulin or low serum IgA | yes |
Hyperinsulinemic hypoglycemia in infancy | no |
Lip pits | no |
Malpositioned kidneys | no |
Idiopathic thrombocytopenia purpura (ITP) | no |
Hypospadias in males | no |
Phenotypic scoring system for KS proposed by the international consensus diagnostic criteria. This table was adapted from the studies of Makrythanasis
Clinical Findings | Possible Score | Scored Features | Feature Present/Number of Points |
---|---|---|---|
Facial features | 0-5 pointsa | abnormal dentition | [–] |
arched eyebrows, sparse lateral one-third | [+] | ||
blue sclerae | [–] | ||
broad nalal root | [+] | ||
averted lower eyelids | [–] | ||
flat nasal tip | [+] | ||
high or cleft palate | [+] | ||
large dysplastic ears | [+] | ||
lip nodules | [–] | ||
long palpebral fissures | [+] | ||
micrognathia | [–] | ||
oligodontia | [–] | ||
ptosis | [–] | ||
strabismus | [–] | ||
thin vermillion of the upper lip and full lower lip | [+] | ||
a 0-3 features = 1 point; 4-6 features = 2 points; 7-9 features = 3 points; 10-12 features = 4 points; 13-15 features = 5 points | |||
Limb/extremity features | up to 1 pointb | brachydactyly or clinodactyly | [+] |
hip dislocation | [–] | ||
lax joints | [–] | ||
persistent fetal pads | [–] | ||
b 0-1 feature = 0 point; 2-4 features = 1 point | |||
Heart | 1 point | 1 | |
Kidney | 1 point | 1 | |
Microcephaly | 1 point | 1 | |
Short stature | 1 point | 1 Noticed during development. | |
Summary | 1-10 points | 3+0+4=7 |
Kabuki syndrome is a rare heterogeneous genetic disorder characterized by typical facial features, of which the most recognizable are elongated palpebral fissures and arched eyebrows giving the children the resemblance to actors in Kabuki theater [14,15]. These features are probably still the most prominent features upon which the clinical diagnosis is reached. Additionally, well defined guidelines were established through past years for better recognition of this rare syndrome [4,13]. Moreover, various combinations of five cardinal manifestations previously mentioned should be taken in consideration by the clinicians when assessing the diagnosis of these patients, because a large number of additional congenital anomalies, together with functional differences, has been reported in individuals pointing to a KS diagnosis [9]. Furthermore, reaching the KS diagnosis in neonates is extremely difficult, as the phenotype seems to evolve during the growth of the child, and typical facial features are not prominent before the age of 3 [4,10]. Therefore, the clinical diagnosis is more focused on the presence of malformations associated with KS, and reevaluations are usually needed before reaching the proper diagnosis, as evidenced in our case [10]. As the KS is a heterogeneous disorder there is phenotypic diversity between the patients, which was observed when phenotypic scoring was applied. Patients carrying the heterozygous pathogenic variant in the
Although our proband is a typical representative of KS (Table 1, Table 2, Figure 2); his diagnosis was reached only after NGS analysis. Due to prenatally observed CHA in our proband, classic karyotyping, QF-PCR, MLPA, and molecular karyotyping was performed in order to exclude aneuploidies, mosaicism, and pathogenic copy number variations (CVNs) during the pregnancy. All the performed test were negative. Additional clinical signs were observed after birth, which indicated the possibility of monogenic disease in our proband, therefore panel NGS sequencing was performed. The described diagnostic pathway is a standard procedure in our laboratory in prenatally and postnatal for observing cases of congenital anomalies.
As mentioned previously, reaching the diagnosis in neonates is still difficult. Our proband thus represents the importance of genotype-phenotype driven NGS analysis in the diagnostic of patients with congenital anomalies.
Congenital heart anomalies, microcephaly, and muscular hypotonia were the most prominent features observed in our proband after birth. Due to the negative results of molecular karyotyping during pregnancy because of observed CHA, an NGS analysis in phenotype-associated genes was performed. A frameshift deletion in exon 39 in the
Pathogenic mutations in the
The
According to the ACMG guidelines [12], heterozygous frameshift deletion in the
According to Xin
In our proband, the CHA observed prenatally is manifested as a consequence of the detected
Postnatal growth deficiency is also common in KS patients and its cause is uncertain [22]. Schott
Despite the genetic diagnosis achieved, the limitation of this study should be emphasized. The use of panel NGS sequencing and then phenotype driven analysis, unable us to discover aberrations in potentially previously unknown Kabuki-associated genes as well as identification other pathogenic variants in genes that were not included in panel but could contribute to proband’s phenotype. However, this type of analysis reduces the possibility of identification of incidental findings as well as identifications of single nucleotide variants in biologically poorly studied genes. However, in this study, a panel that enriches exons of 4813 genes with clinical relevance to the disease was used, leading to the identification of a
In conclusion, while KS is a well-characterized genetic disorder, placing a diagnosis in infants is still challenging. Our proband demonstrated that careful phenotyping of the patients that undergo NGS testing is needed for the diagnostics to be successful. It also emphasizes the benefit of NGS analysis in early diagnostics, resulting in prospect of target therapies and improving the ability to monitor disease progression [2,4,9].