Cornelia de Lange syndrome (CdLS, OMIM #122470, 300590, 300882, 610759, 614701) is a multisystem disorder that causes diverse physical and neurocognitive problems [1]. The prevalence is estimated to be approximately 1 in 10,000-30,000 live births worldwide, irrespective of the ethnic group [1,2]. CdLS is inherited in either an autosomal dominant or X-linked manner depending on the affected genes, and the majority of the cases are sporadic [3]. Common clinical features of CdLS include a distinctive craniofacial appearance, growth failure, developmental delay/intellectual disability, upper limb abnormalities, and hypertrichosis [1]. Although pediatricians can easily recognize patients with a typical CdLS phenotype, arriving at the diagnosis is often complicated because of the clinical overlap with other disorders showing CdLS-like phenotypes, such as CHOPS syndrome, KBG syndrome, or Rubinstein-Taybi syndrome [1,2].

Pathogenic variations of the genes belonging to the cohesion pathway cause CdLS and are considered to be a group of ‘cohesinopathy’ [4]. Cohesin plays an important role in chromatid cohesion, gene expression, and DNA repair [5]. There are five CdLS-causative genes that are involved with the cohesin complex: three core cohesion subunits (SMC1A, SMC3, and RAD21) and two cohesion-regulatory proteins (NIPBL and HDAC8) [2,6]. Pathogenic variants of NIPBL (5p13.2, MIM 608667) are the most common genetic causes, accounting for approximately 60–70% of patients with CdLS [1,2]. In addition, 5-10% of patients with CdLS carry pathogenic variants of SMC1A (Xp11.22, MIM 300040), HDAC8 (Xq13.1, MIM 300882), SMC3 (10q25.2, MIM 606062), and RAD21 (8q24.11, MIM 614701) [1,2].

Genotype-phenotype correlation has been reported in patients with CdLS. Patients with NIPBL variants usually present the most distinctive clinical features, followed by those with variants of HDAC8, RAD21, SMC1A, and SMC3 [6]. In addition, truncating pathogenic variants of NIPBL result in more distinctive phenotypes compared with those carrying other genetic variants (such as, missense variants of NIPBL or pathogenetic variants of SMC1A, SMC3, RAD21) [2]. In Korea, 16 genetically confirmed cases of CdLS (10 sporadic, 6 familial from two families) have been reported so far, but the reported variants were limited to either NIPBL or SMC1A [7-12]. The clinical spectrum of CdLS as a cohesinopathy in Korean patients with CdLS is largely unknown. Hence, in the present study, we investigated the clinical and molecular characteristics of 15 Korean patients with CdLS and evaluated their genotype-phenotype correlations.

1. Patients

Fifteen unrelated patients who had shown suspicious features of CdLS with a clinical score (CS) of 4 or more at the Seoul National University Hospital between November 2010 and June 2021 were enrolled in this study. The medical records of these patients were retrospectively reviewed. Standard deviation scores (SDS) for growth parameters, including height, weight, and head circumference, were assigned in each case both at birth based on the Fenton growth charts and postnatally based on the 2017 Korean National Growth Charts [13,14]. Prenatal and postnatal growth retardation were defined as a birthweight and height –2 SDS below the scores of the respective age- and sex-matched controls [15,16]. Microcephaly was defined as a head circumference –2 SDS below the scores of the corresponding age- and sex-matched population group [17]. Developmental milestones were evaluated by pediatric neurologists, and analysis of the brain magnetic resonance imaging (MRI) was performed by neuroradiologists. The full-scale intelligence quotient (FSIQ) was assessed in three patients using the Korean-Wechsler Preschool and Primary Scale of Intelligence-Fourth edition or Korean-Leiter International Performance Scale-Revised. Eye and ear-nose-throat abnormalities were ascertained by evaluating the ophthalmology and otorhinolaryngology records, respectively. The combined cardiac and renal malformations were assessed by performing echocardiography and renal sonography.

The CS was calculated for each patient according to the consensus statement of CdLS [1]: 2 points were allotted for each of the six cardinal features (synophrys and/or thick eyebrows, short nose, concave nasal ridge and/or upturned nasal tip, long and/or smooth philtrum, thin upper lip vermilion and/or downturned corners of mouth, hand oligodactyly and/or adactyly, and congenital diaphragmatic hernia), and 1 point was allotted for each of the seven suggestive features (global developmental delay and/or intellectual disability, prenatal growth retardation, postnatal growth retardation, microcephaly, small hands and/or feet, short fifth finger, and hirsutism). Patients with a CS ≥11 (with at least three cardinal features) were classified as having classic CdLS. Patients with CS of 9-10 points (with at least two cardinal features) were considered to have non-classic CdLS, whereas those with CS of 4-8 points (with at least 1 cardinal feature) were defined as other phenotypes sharing limited signs of CdLS [1]. This study was approved by the Institutional Review Board of the Seoul National University Hospital (IRB no. 2106-068-1225) and exempt from written informed consent.

2. Molecular genetic analysis

Genomic DNA had been extracted from the leukocytes in the peripheral blood using a DNA isolation kit (QIAGEN, Hilden, Germany). Sanger sequencing had been performed to identify the NIPBL pathogenic variant in two patients with the classic CdLS phenotype (cases 1 and 2). The coding exons (exons 2-47) and flanking intronic sequences of NIPBL (reference sequence, NM_133433) had been screened using polymerase chain reaction amplification and bidirectional direct sequencing. Five patients (cases 4, 7, 9, 12, and 14) had undergone targeted next-generation sequencing for CdLS, which analyzed five genes (NIPBL, SMC1A, SMC3, HDAC8, and RAD21). The remaining eight patients had undergone whole-exome sequencing (WES) using a HiSeq 2500 sequencing system (Illumina Inc., San Diego, CA, USA). All the sequence variants had been classified according to the standards and guidelines set by the American College of Medical Genetics and Genomics (ACMG) [18]. Pathogenic or likely pathogenic variants had been defined as causative variants, and if possible, the clinically relevant variants had been verified using Sanger sequencing of the parental samples. The DNA from one patient (case 12) without any meaningful variants in the exome analysis had been additionally subjected to a single-nucleotide polymorphism microarray analysis using Affymetrix CytoScan 750 K Array (Affymetrix, Santa Clara, CA, USA).

3. Statistics analysis

Continuous variables with a normal distribution are expressed as mean values with standard deviation, whereas those without a normal distribution are expressed as median values with range. All categorical variables are presented as numbers and percentages of the participants. The comparison between NIPBL group and non-NIPBL group was performed using a Student’s t-test and Mann–Whitney U-test for continuous variables, and Fisher’s exact test for analyzing the categorical variables. Group differences according to the four genes involved (NIPBL, SMC1A, SMC3, and HDAC8) were analyzed using the Kruskal–Wallis test. A P-value less than 0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics for Windows (version 25.0; IBM Co., Armonk, NY, USA).

1. Baseline characteristics

Table 1 shows the baseline clinical characteristics of the 15 Korean patients with CdLS (3 males and 12 females). The median age at the time of diagnosis was 1.7 (range, 0.0-11.8) years, and median follow-up duration was 3.8 (range, 0.4-11.7) years. The mean birthweight SDS was –1.8±0.9, and the mean SDS of height, weight, and head circumference at the first visit (at a mean age of 1.3±1.5 years) were –2.4±2.3, –2.1±1.6, and –2.8±1.3, respectively. Eight (53.3%) patients were classified as having classic CdLS, two (13.3%) as having non-classic CdLS, and five (33.3%) as having other phenotypes sharing limited signs of CdLS. Regarding cardinal features, synophrys and/or thick eyebrows were most frequently noted in 13 (86.7%) patients, followed by a thin upper lip vermilion and/or downturned corners of the mouth in 12 (80.0%), short nose in 11 (73.3%), long and/or smooth philtrum in 10 (66.7%), and congenital diaphragmatic hernia in one (6.7%) patient. Regarding suggestive features, global developmental delay and/or intellectual disability was noted in 14 (93.3%) patients (except case 9, due to being lost to follow-up at 6 months of age), followed by microcephaly in 12 (80.0%), postnatal growth retardation in 11 (73.3%), hirsutism in 10 (66.7%), small hands and/or feet in 8 (53.3%), prenatal growth retardation in 6 (40.0%), and a short fifth finger in 3 (20.0%) patients (Fig. 1). Regarding other clinical features, ophthalmologic abnormalities including ptosis, myopia, hypermetropia, amblyopia, and/or strabismus were observed in ten (66.7%) patients, followed by hearing impairments in seven (46.7%), gastrointestinal abnormalities including gastroesophageal reflux disease in six (40.0%), cardiac anomalies in four (26.7%), and genitourinary abnormalities including small kidney, vesicoureteral reflux, and/or undescended testis in four (26.7%) patients.

2. Clinical outcomes

The mean SDS of height, weight, and head circumference at last visit (at a mean age of 4.8±3.0 years) were –3.0±2.3, –2.8±2.1, and –3.4±1.9, respectively. The responses to growth hormone (GH) in two GH-treated patients were insufficient; the first-year changes in height SDS were –1.4 in case 4 (small for the gestational age without catch-up growth), and +0.2 in case 14 (GH-deficient). Four (26.7%) patients (case 6, 7, 8, and 10) developed seizures at the median age of 1.8 (range, 0.1-4.3) years. Among the 10 patients who underwent brain MRI, six showed abnormal findings: simplified gyral patterns (cases 2, 6, and 8), retrocerebellar cyst (case 7), diffuse brain atrophy (case 15), and pituitary gland hypoplasia (case 14 with GH deficiency). The FSIQ scores were below 40, 40, and 47 in cases 2, 3, and 4, respectively.

3. Molecular genetic characteristics

Fifteen causative variants (including 12 novel variants) were identified: NIPBL in five (33.3%, including three males), SMC1A in three (20.0%), SMC3 in three (20.0%), and HDAC8 in four (26.7%) cases (Table 2). Novel genetic variations included one 458Kb microdeletion of Xq13.1q13.2, which included HDAC8 (Hg19, chromosome X:71502595-71960802) and 11 sequence variants: four NIPBL (c.5428-1G>T, c.2884_2885delAA, c.3G>A, and c.2843G>T), one SMC1A (c.3103C>T), three SMC3 (c.491G>A, c.3476-2A>G, and c.2523_2525delCCA), and three HDAC8 variants (c.149delT, c.437+1G>C, and c.193G>T). Among them, eight novel sequence variants were considered causative variants as they caused frameshift, nonsense, and start-loss mutations, leading to premature termination of translation or mis-splicing, which led to the formation of alternative transcripts. According to ACMG criteria, two novel missense variants, c.2843G>T (p.Gly948Val) in NIPBL and c.3103C>T (p.Gly164Asp) in SMC3, were considered as likely pathogenic and pathogenic variants (PM1+PM2+PM6+PP3+PP4 and PVS1+PS2+PM2+PP3, respectively). In all the 12 affected probands with parental samples available, the disorder was caused by a de novo pathogenic variant in both autosomal dominant CdLS (NIPBL and SMC3) and X-linked CdLS (SMC1A and HDAC8).

4. Genotype-phenotype correlations

All five individuals with NIPBL variants showed classic CdLS phenotypes without significant differences in CS according to variants-type (15.0 for loss-of-function variants vs. 13.0 for missense variants, P=0.075). Out of the three females with SMC1A variants, one (case 6) had classic CdLS, one (case 8) had non-classic CdLS, and one (case 7) had a phenotype sharing limited signs of CdLS. Out of the three females with SMC3 variants, one (case 11) had a classic CdLS phenotype and two (cases 9 and 10) had limited signs of CdLS. Of the four females with HDAC8 variants, classic CdLS phenotype was present in one case (cases 13), non-classic CdLS phenotype in another one case (case 14), and there were limited signs of CdLS in the other two cases (cases 12 and 15) (Fig. 2). The CSs were highly variable and ranged from 5 to 16: mean CS of 14.2 in NIPBL, 9.3 in SMC1A, 8.8 in HDAC8, and 8.0 in SMC3 in the order with significant intergroup difference between NIPBL and SMC3 (P=0.029). A comparison between NIPBL group and non-NIPBL group revealed a significant female predominance in the non-NIPBL group compared to that in NIPBL group (40% vs. 100%, P=0.022). In addition, NIPBL group showed a significantly higher CS (14.2±1.3 vs. 8.7±2.9, P<0.001) and a marginally lower height SDS at the first visit (–4.0±2.6 vs. –1.7±1.7, P=0.052) compared to the scores in the non-NIPBL group (Table 3).

In the present study, we described the clinical and molecular characteristics of 15 Korean patients with CdLS. These patients presented a wide clinical spectrum for CdLS with varying degrees of clinical severity. We identified 15 causative variants (including 12 novel variants) in four genes (NIPBL, SMC1A, SMC3, and HDAC8). Patients carrying NIPBL variants had a more distinctive phenotype than those carrying variants of other cohesion complex genes (SMC1A, SMC3, and HDAC8).

The common characteristics presented were consistent with those previously reported: typical facial features, developmental delay and/or intellectual disability, microcephaly, and prenatal and postnatal growth retardation [1,19-21]. Distinctive facial appearance (such as synophrys, thick eyebrows, thin upper vermilion border of the lip, downturned corners of the mouth, short nose, and long and smooth philtrum), as reported in previous studies, were the most recognizable clinical findings in these patients, especially in those with the classic CdLS phenotype [19,22]. Prenatal and postnatal growth retardation is a hallmark of CdLS, and the mean adult height was 155.8 cm in men and 131.1 cm in women among clinically diagnosed CdLS individuals [23]. GH secretion is generally normal in most children diagnosed with CdLS [24-27]. However, the GH response in our two GH-treated patients (including one with GH-deficient) was not sufficient, although an appropriate response has been reported in a mildly affected case without GH deficiency [28]. Regarding neurological findings other than developmental delay, seizures developed in 26.7% of our patients. Focal seizures were observed in three out of four patients and 1 to 5 kinds of antiepileptic drugs were necessary to control seizures, which were in accord with previous reports [3,29]. Abnormal neuroimaging findings included microcephaly, gyral simplification, and diffuse brain atrophy, which have also been reported as common features in patients with CdLS [30]. The degree of limb involvement in our CdLS cohort was mild (small hands and feet only) in contrast to 25-46% patients having major limb anomalies (such as oligodactyly and adactyly) in previous reports [3,21,31]. Compared to the reports showing the clinical features of CdLS patients from China and Asia, Korean patients showed a similar distribution of typical characteristics except for higher rate of hearing loss (46.7% in Korean vs. 6.7% in Chinese or 22.2% in Asian) [19,20]. Clinodactyly was not significant in Korean patients compared to Chinese and Asian patients (0% in Korean vs. 66.7% in Chinese or 55.6% in Asian) [19,20].

Patients with SMC1A and SMC3 variants showed less distinctive craniofacial appearance and milder prenatal growth retardation compared to those with NIPBL variants [32-34]. Also, patients with SMC1A variants had milder postnatal growth retardation with higher rate of seizure and self-injurious behavior resembling Rett syndrome [32,33]. Congenital heart diseases were less frequent in patients with SMC3 variants and were similar to previous reports [34].

We identified 15 genetic causative variants of NIPBL (33.3%), SMC1A (20.0%), SMC3 (20.0%), and HDAC8 (26.7%) in Korean patients confirmed to have CdLS. Of these, 12 causative variants were identified using a multigene panel for CdLS or WES sequencing in accordance with current recommendations [1]. As more information has come to light regarding the molecular basis of CdLS, and as the symptomatic features presented by the affected individuals are milder or atypical, next-generation sequencing-based screening (either gene panel or WES, which contains the currently known CdLS genes) is recommended as the first-line molecular testing method to diagnose CdLS [1]. The variants of NIPBL and SMC1A genes reported in our cohort were different from any of the previously reported variants in Korean patients (Supplementary Table 1) [7-12]. Given that most variants of CdLS are de novo, they are commonly present as private pathogenic variants [3]. In our cohort, all seven patients with X-linked CdLS (SMC1A and HDAC8) were female. The severity in female carriers of typical X-linked genes, including HDAC8, is largely dependent on the pattern of X-inactivation. However, SMC1A escapes X-chromosome inactivation, and a dominant-negative effect in a carrier female results in a milder phenotype than in a male patient with the same variant [1,35].

Among the CdLS causative variants identified in our study, c.6893G>A of NIPBL (in case 5 with classic CdLS, CS of 13) has been previously reported in individuals with CdLS: two probands showed growth retardation of 25-75th percentile on the CdLS growth curves and developmental delay of more than 2 years behind normal developmental standards [23,36], a male showed mild mental retardation, radial dislocation, and pulmonary stenosis [37], and two probands showed nasolacrimal duct obstruction and myopia (astigmatism and microphthalmia in one) as ophthalmologic findings [38]. The c.3285+1G>C variant in SMC1A (in case 6 with classic CdLS, CS of 13) has also been reported in a female with growth and developmental delay, micro- and holoprosencephaly, and spina bifida [39]. On the other hand, the c.2611C>T of SMC1A (in case 7 with limited CdLS signs, CS of 5) was characterized on two separate occasions as a condition related to congenital muscular hypertrophy-cerebral syndrome and as intellectual disability [40]. This phenotypic variability associated with identical variants implies that other genetic and/or environmental factors may modify the clinical features [36]. In our study, patients carrying NIPBL variants showed more distinctive clinical features than those carrying other variants (SMC1A, SMC3, HDAC8), which was consistent with previous reports [6]. In addition, haploinsufficiency of NIPBL has been suggested to result in a more distinctive phenotype in loss-of-function NIPBL variants than in missense or in-frame deletion NIPBL variants [3,6,36]. However, the differences according to variant type in the NIPBL group were not significant in our study, largely due to the small sample size in each subgroup.

Our study had several limitations. First, there was a possibility of selection bias, as our study was performed in a single tertiary center. Second, statistical significance could not be estimated for some parameters related to genotype-phenotype correlation due to the small sample size. Third, due to the retrospective study design and short follow-up period, we could not evaluate the long-term growth and developmental outcomes. In addition, the objective degree of intellectual disability (such as the FSIQ) was measured in only a limited number of patients. However, our study was strengthened by detailed phenotyping of a Korean CdLS cohort with molecular confirmation. In addition, we identified 15 causal variants, of which 12 were novel, in genes involved in molecular pathways underlying cohesinopathies and some genotype-phenotype correlations.

In conclusion, we identified the clinical and genetic heterogeneity in Korean patients with CdLS. The clinical severity was higher in patients with NIPBL variants than in those carrying other variants (SMC1A, SMC3, HDAC8). Further studies are needed to elucidate the exact pathomechanisms and phenotypic consequences of cohesinopathy on the CdLS spectrum.

The authors declare that they do not have any conflicts of interest.

Conception and design: JMK. Acquisition of data: DK, HYK, JHC. Analysis and interpretation of data: DK, HYK, JHC. Drafting the article: DK. Critical revision of the article: HYK, JMK. Final approval of the version to be published: JMK.