Introduction
Both 6q trisomy and 2q monosomy are rare chromosome abnormalities. Diagnosing genome structural variations (SVs) remains challenging due to the complexity of larger chromosomal events, the complete range of SVs, and the need for appropriate sequencing techniques [
1]. A thorough evaluation of all types of SVs could potentially elucidate the diverse clinical manifestations, which may be attributed to deletions of varying sizes in different genes. In this study, we successfully employed a combination of exome and genome sequencing techniques to confirm a diagnosis of pathogenic SV involving an unbalanced translocation between chromosomes 2q and 6q in a family.
The proband initially came to our hospital seeking treatment for ptosis. During the ophthalmic examination, several other concerning features were identified, including downward-slanted palpebral fissures and bilateral optic nerve hypoplasia. Additionally, she exhibited a prominent forehead, midface hypoplasia, hypertelorism, a flat nasal bridge, a short nose, a small mouth with thin lips, micrognathia, and blepharophimosis. She also experienced postnatal growth deficits and developed severe intellectual disability and was affected by significant neurological and skeletal dysmorphism anomalies. This is the second reported case of a patient simultaneously carrying partial monosomy 2q and partial trisomy 6q. In a previous report, a patient carrying the karyotype of 46,XY,der(2)t(2;6)(q37.3;q26) presented intellectual disability, obesity, brachydactyly, and short stature. The author identified the 2q deletion as the primary driver of the phenotype due to AHO-like syndrome (MIM # 600,430) [
2]. In comparison with the previous case, our patient presented with a more severe form of congenital cranial dysinnervation disorder (CCDD) (MIM # 620,469), along with abnormal thorax development. This expands the range of phenotypic manifestations associated with the unbalanced translocation between the distal regions of chromosomes 2q and 6q. Her karyotype showed an unbalanced translocation of 46,XX,del(2)t(2;6)(q37.1;q25.3). This variant was transmitted from her unaffected father [46,XY,t(2;6)(q37.1;q25.3)], which led to del(2)(q37.1;37.3) and dup(6)(q25.3;q27). We also obtained the precise breakpoint of a de novo heterozygous copy number deletion [del(2)(q37.1q37.3)chr2:g.232963568_24305260del] and a copy number duplication [dup(6)(q25.3q27)chr6:g.158730978_170930050dup].
The accurate molecular diagnosis of genetic disorders makes it possible to better understand the pathogenesis of complex syndromes, which is imperative to families with a history of diseases and couples with consecutive abnormal fetuses showing similar phenotypes. Identifying pathogenic variants is an excellent method for explaining the correlation between phenotype and genotype and the possible effects on biological mechanisms.
Discussion
Here, we report a proband with characteristic features of the clinical syndrome, including downward-slanting palpebral fissures; delayed development of bilateral optic nerve hypoplasia; facial deformity with a small mouth, thin lips, micrognathia, and blepharophimosis; short neck; abnormal deciduous teeth; and malformations of the thorax and short ribs. The proband showed severe CCDD and mental and developmental disability. To our knowledge, this is the second report that depicts the features of partial 6q trisomy and partial monosomy 2q with translocation. The duplicated region in our patient, 6q25.3-q27, includes 53 protein-coding genes (49 OMIM genes), and the deleted region, 2q37.1-q37.3, includes 85 protein-coding genes (74 OMIM genes).
In a previous report, the proband was described as carrying 46,XY,der(2)t(2;6)(q37.3;q26), along with a balanced translocation [t(2;6)] in his father and sister [
2]. This chromosomal abnormality is associated with AHO-like syndrome, also known as brachydactyly mental retardation syndrome (BDMR syndrome). This syndrome is characterized by several features, including brachydactyly type E (48%), overweight and obesity (34%), cognitive-behavioral issues (79%), and dysmorphic craniofacial or skeletal dysmorphism (86%) [
12]. The main contributing factor to the proband’s phenotype was believed to be monosomy of the 2q37.3-qter region. Patients with chromosome 2q37 deletion syndrome exhibit a wide range of clinical manifestations, which can be attributed to the varying length and deletion of specific genes in this region. Among the OMIM morbid genes, several genes have been identified as candidates correlated with the phenotype of our proband’s 2q37 deletion.
COL6A3 (MIM *120,250), which encodes type IV collagen, has been associated with short stature, obesity, and brachymetaphalangia in affected individuals [
13].
PDCD1 (MIM *600,244), which codes for a cell surface membrane protein, has been reported to be associated with malformation, as it plays a role in programmed cell death or apoptosis in embryogenesis [
14]. The
HDAC4 (MIM *605,314) gene, which is involved in chromosomal packaging, has been implicated in the major phenotypes of BDMR syndrome through its ability to repress transcription [
12].
An imbalance in gene dosage can have deleterious effects, with deletions typically causing a more severe clinical presentation than duplications [
15]. In addition to BDMR, our proband also showed severe CCDD, which manifested as downward-slanting palpebral fissures and delayed development of bilateral optic nerve hypoplasia. These symptoms indicate central nervous system abnormalities and ocular defects, suggesting a partial role of trisomy 6q. Both trisomy 6q and monosomy 2q are rare chromosome abnormalities. Trisomy 6q is associated with severe physical and intellectual disability, hypertelorism, feeding difficulties, and dysmorphic features (microcephaly, acrocephaly, prominent forehead, flat nasal bridge, downward-slanting palpebral fissures, carp mouth, micrognathia, short webbed neck, club feet, and flexion deformity) [
16,
17]. While both trisomy 6q and monosomy 2q37 commonly exhibit signs of psychomotor retardation, growth retardation, thin upper lip, prominent forehead, flat nasal bridge, micrognathia, and deformities of hand and foot (Table
3), trisomy 6q appears to be more associated with severe neurological abnormities, whereas monosomy 2q is more linked to skeletal abnormities along with mild neurological abnormities. Among the genes in the duplicated region of our proband, the
DLL1 (MIM *606,582) gene is involved in neurodevelopmental disorders with nonspecific brain abnormalities in the trisomy 6q region. The
DLL1 gene encodes a Notch ligand and is essential for developing the nervous system and somites [
18]. Additionally, the
PSMB1 (MIM *602,017) gene is responsible for the breakdown of intracellular proteins, and its malfunction can lead to neurodevelopmental disorders characterized by microcephaly, hypotonia, and absent language [
19]. Our proband presented with severe skeletal dysmorphism abnormalities that are more closely associated with monosomy 2q. It appears that both monosomy 2q and trisomy 6q contribute to the complex phenotype of our proband. Partial duplication of the distal long arm of chromosome 6 alone can result in a severe phenotype [
16]. This is commonly accompanied by severe intellectual disability, hallmark facial malformation, short stature, and severe motor abnormalities. Compared with pure monosomy, the phenotypic difference was believed to be due to a larger trisomic region exerting the effect of deletion when deletion and duplication occurred in the same patient.
The accurate molecular diagnosis of genetic disorders makes it possible to better understand the pathogenesis of complex syndromes, which is imperative to families with a history of diseases and couples with consecutive abnormal fetuses showing similar phenotypes. It is necessary to accurately predict the features (copy, content, and structure) of SV discovery and genotyping. However, it remains a challenge to find a single method or technology that can comprehensively detect all SVs within a genome [
1]. A single method can neither provide a definite determination of the breakpoint nor reveal the location and orientation. The combination of exome and genome sequencing techniques is the optimum selection to detect the precise site of SVs. Exome and genome sequencing are proposed to be applied to pediatric patients with congenital anomalies or intellectual disabilities [
20]. The genome sequencing approach is a complementary method to locate the accurate site of the SVs when the WES technique and the phenotype point to a pathological CNV as the leading cause. Thus, exome sequencing rapidly located the pathological CNVs, even though the CNV information is obscure. OGM and ONT can efficiently complement exome sequencing to locate the breakpoint of SVs, identify the leading cause and have the potential to uncover more complex genomic structures that are missed by low-resolution methods. Although the long-read technique takes advantage of other techniques in detecting SVs [
1], it is unlikely that, in our case, these variants could be identified from long-read data alone without prior knowledge of the region of interest.
In the present study, we highlight the role of combining exome and genome sequencing techniques in resolving the SVs with a translocation between 2q and 6q in one family. In this study, we initially utilized WES to identify the pathological CNVs on chromosomes 2q and 6q but failed to detect the precise location. WES is a commonly used, high-throughput short-read technology for detecting exact breakpoints and has resulted in dramatic increases in novel gene discovery for Mendelian disorders. It initially used a hybridization microarray approach and has been successful in identifying regions and distinguishing alleles with high sensitivity. Nevertheless, WES has limitations in capturing larger genomic variants (CNVs, ≥ 50 bp) and cannot detect SVs reliably due to the technical and analytical challenges around identifying and interpreting SVs that are associated with deletions or duplications and could be overlooked throughout the full scope of the genome. Additionally, breakpoints located within repetitive regions of the genome are unmappable by WES, which means a lower sensitivity. In addition, balanced translocations cannot be detected since they do not alter chromosome copy numbers. Therefore, not surprisingly, WES only provided results that pointed to a pathological CNV with a deletion at the terminal part of 2q37 and a duplication of 6q that may be the leading cause, but without precise resolution of breakpoints. Therefore, we tried other genome sequencing methods.
With the development of next-generation sequencing, ChIP-Seq allows for the sequencing of DNA fragments rather than hybridizing them to an array, making it possible to develop a robust statistical model that describes the complete analysis procedure and allows the computation of essential confidence values for the detection of CNVs. This has led to CNV sequencing as a promising technique. Low-resolution CNV-seq is a short-read whole-genome sequencing method that has proven particularly powerful in detecting SVs, variants in GC-rich regions, and variants in noncoding regulatory regions [
21]. We further used CNV-seq to identify the pathogenic CNVs; CNV-seq rapidly identified the existence of the pathogenic CNV and classified the abnormality as a 12.01 Mbp copy number duplication of distal 6q25.3-q27 and 9.32 Mbp copy number deletion of distal 2q37.1- q37.3. However, there are short-read technical challenges in resolving the exact structures of SVs given their substantial diversity and proximity to repetitive regions [
22]. This method could locate the sequence but could not detect the existence of the translocation.
When considering the two involved chromosomes, we applied the long-read technique to identify the precise site and possible origin. The long-read genome sequencing methods OGM and ONT also made it possible to map the breakpoint precisely to the region covered by 2q and 6q [
23]. Based on nanochannel-based genome mapping technology, OGM utilizes enzymes to label high-molecular-weight DNA without breaking the DNA or polymerase chain reaction (PCR), and fluorescently tagged mega-base size DNA is obtained. These long DNA fragments are linearized through pillars and imaged in nanochannels as a long stretch of single-stranded DNA that passes through a protein nanopore that is stabilized in an electrically resistant polymer membrane [
24]. By applying a voltage across this membrane, sensors detect the ionic current changes caused by nucleotides occupying the pore in real time as the DNA molecule passes through. Although it cannot offer base-level sequence information, it can visualize DNA structure and reveal a wide spectrum of SVs. Nanopore sequencing is another technique that directly detects the input molecule without DNA amplification or synthesis; there is no apparent limit to the length of DNA that can be sequenced [
25]. Long-read techniques have the advantage of reads of 10–100 kb, allowing for more accurate mapping, particularly over repetitive regions, and facilitating phasing [
26]. We obtained the precise breakpoint of a de novo heterozygous copy number deletion [del(2)(q37.1q37.3)chr2:g.232963568_24305260del] and a copy number duplication [dup(6)(q25.3q27)chr6:g.158730978_170930050dup], and the parental balanced translocation was apparent. However, unlike the limits of CNV-seq in balanced translocation, long reads can locate the causative region of balanced and unbalanced translocations, but CNV-seq is much more cost-effective. A combination of karyotyping and CNV-seq is another suggested approach for the diagnosis of submicroscopic unbalanced genomic rearrangements [
27]. Although karyotyping is a routine standard cytogenetic method to detect SVs, its drawbacks are low resolution and the inability to indicate the genomic localization and orientation of duplicated segments or insertions (CNV microarrays). It usually takes much longer to obtain sequencing information. Therefore, the combination of exome and genome sequencing is a more efficient choice.
This study typified a pathological CNV offspring with unbalanced translocation caused by parental balanced translocation. Approximately one in every 300–500 individuals has a balanced reciprocal translocation according to varied estimates [
28]. Balanced translocations can take the form of inversions or translocations and lead to CNVs, which often involve changes in DNA dosage. CNVs, partial chromosomal deletions and duplications are significant contributors to the genome variability among individuals and can either be pathogenic or have no clinical consequences. A diverse set of rearrangements involving the exchange of segments between chromosomes is common in humans. Most balanced translocation carriers have no phenotypic consequences but have a higher risk of infertility, miscarriage, and unbalanced progeny. Moreover, their offspring may have a continuous spectrum of phenotypic effects of pathogenic CNVs, from adaptive traits to underlying causes of disease to embryonic lethality risk of an associated reciprocal translocation [
29]. Translocations have also been productive in identifying new candidate genes underlying common clinical phenotypes that may arise from dysfunction of any number of genes, as in alveolar soft-part sarcoma [
30], acute myeloid leukemia [
31], and autism [
32]. Researchers are struggling to identify a rapid, cost-effective solution with a low per-base error rate to assess this issue. This solution could provide new information to study translocation formation and may suggest ways to prevent its occurrence. Thus, exome and genome sequencing techniques could be applied to resolve chromosomal balanced translocation and complex SVs. This combination ensures the accurate determination of variants. Considering the cost, time, and throughput, the combination of WES and CNV-seq may be a good alternative for SV detection but cannot directly detect balanced translocations; thus, other combinations should be taken into consideration.
In conclusion, the combination of exome and genome sequencing techniques is suggested to secure precise breakpoints of the location of SVs, which could be in the form of CNVs or translocations, and provide evidence of an irregular phenotype due to the translocation between 6q + and 2q-. The genome sequencing techniques could be CNV-seq, ONT, or OGM, with CNV-seq being much more cost-effective than the other two techniques. Our study also emphasizes that accurate molecular diagnosis of genetic disorders is critical to interpreting the pathogenesis of complex syndromes and supplying evidence of the formation of replication-based mechanisms for complex structural variations, which is imperative to families with a history of diseases and couples with consecutive abnormal cases showing similar phenotypes. It is also helpful for the patient’s mother to undergo prenatal examination in the event of future pregnancies.
Table 3
Comparison of the phenotype between the 2q- and 6q + syndromes
Psychomotor retardation | + | + | + | + |
Growth retardation | + | + | + | |
Microcephaly | | + | | |
Broad/round face | + | | | |
Prominent forehead | + | + | | + |
Flat nasal bridge | + | + | + | |
Downward-slanting palpebral fissures | | + | + | |
Short palpebral fissures | + | | | |
High arched palate | | + | | + |
Carp/bow shaped mouth/Microstomia | | + | + | |
Thin upper lip | + | + | + | + |
Micrognathia | + | + | + | |
Short webbed neck | | + | + | + |
Joint contractures | | + | + | |
Camptodactyly | + | + | + | |
Finger ulnar deviation/syndactyly | + | + | | |
Small feet | + | + | + | + |
Clubfoot | | + | | |
Obesity | + | | | + |
Table 4
The genes in the CNVs with OMIM numbers
chr2:g.232963568_24305260del | AGXT | #259900 | *604285 | Hyperoxaluria, primary, type 1 |
ATG16L1 | #611081 | *610767 | Inflammatory bowel disease (Crohn disease) 10 |
CAPN10 | #601283 | *605286 | Diabetes mellitus, noninsulin-dependent 1 |
COL6A3 | #158810 | *120250 | Bethlem myopathy 1 |
| #616411 | *120250 | Dystonia 27 |
| #254090 | *120250 | Ullrich congenital muscular dystrophy 1 |
D2HGDH | #600721 | *609186 | D-2-hydroxyglutaric aciduria |
KIF1A | #614255 | *601255 | NESCAV syndrome |
| #614213 | *601255 | Neuropathy, hereditary sensory, type IIC |
| #610357 | *601255 | Spastic paraplegia 30, autosomal dominant /recessive |
MLPH | #609227 | *606526 | Griscelli syndrome, type 3 |
NDUFA10 | #618243 | *603835 | Mitochondrial complex I deficiency, nuclear type 22 |
PDCD1 | #126200 | *600244 | Multiple sclerosis, disease progression, modifier of |
| #605218 | *600244 | Systemic lupus erythematosus, susceptibility to, 2 |
PER2 | #604348 | *603426 | ?Advanced sleep phase syndrome, familial, 1 |
SAG | #258100 | *181031 | Oguchi disease-1 |
| #613758 | *181031 | Retinitis pigmentosa 47, autosomal recessive |
| #620228 | *181031 | Retinitis pigmentosa 96, autosomal dominant |
TRAF3IP1 | #616629 | *607380 | Senior-Loken syndrome 9 |
TWIST2 | #200110 | *607556 | Ablepharon-macrostomia syndrome |
| #209885 | *607556 | Barber-Say syndrome |
| #227260 | *607556 | Focal facial dermal dysplasia 3, Setleis type |
| UGT1A1 | #218800 | *191740 | Crigler-Najjar syndrome, type I |
| | #606785 | *191740 | Crigler-Najjar syndrome, type II |
| | #237900 | *191740 | Hyperbilirubinemia, familial transient neonatal |
| | #601816 | *191740 | Bilirubin, serum level of, QTL1 |
| | #143500 | *191740 | Gilbert syndrome |
| DTYMK | #619847 | *188345 | Neurodegeneration, childhood-onset, with progressive microcephaly |
| HDAC4 | #619797 | *605314 | Neurodevelopmental disorder with central hypotonia and dysmorphic facies |
| ACKR3 | #619215 | *610376 | ?Oculomotor-abducens synkinesis |
chr6:g.158730978_170930050dup | DLL1 | #618709 | *606582 | Neurodevelopmental disorder with nonspecific brain abnormalities and with or without seizures |
ERMARD | # 615544 | 615532 | ?Periventricular nodular heterotopia 6 |
IGF2R | #114550 | *147280 | Hepatocellular carcinoma, somatic |
LPA | #618807 | *618807 | LPA deficiency, congenital; Coronary artery disease, susceptibility to |
MPC1 | #614741 | *614738 | Mitochondrial pyruvate carrier deficiency |
PDE10A | # 616921 | *610652 | Dyskinesia, limb and orofacial, infantile-onset |
| #616922 | *610652 | Striatal degeneration, autosomal dominant |
PLG | #619360 | *173350 | Angioedema, hereditary, 4 |
| #217090 | *173350 | Plasminogen deficiency, type I ;Dysplasminogenemia |
PRKN | #211980 | *602544 | Adenocarcinoma of lung, somatic |
| #167000 | *602544 | Ovarian cancer, somatic |
| #600116 | *602544 | Parkinson disease, juvenile, type 2 |
RNASET2 | # 612951 | *612944 | Leukoencephalopathy, cystic, without megalencephaly |
RSPH3 | #616481 | *615876 | Ciliary dyskinesia, primary, 32 |
SMOC2 | #125400 | *607223 | Dentin dysplasia, type I, with microdontia and misshapen teeth |
SOD2 | #612634 | *147460 | Microvascular complications of diabetes 6 |
TBP | #607136 | *600075 | Spinocerebellar ataxia 17 |
| #168600 | *600075 | Parkinson disease, susceptibility to |
TBXT | #615709 | *601397 | Sacral agenesis with vertebral anomalies |
| #182940 | *601397 | Neural tube defects, susceptibility to |
THBS2 | #603932 | *603932 | Lumbar disc herniation, susceptibility to |
ACAT2 | 614055 | *100678 | ?ACAT2 deficiency |
PNLDC1 | #619528 | *619529 | Spermatogenic failure 57 |
CEP43 | *605392 | | Myeloproliferative disorder |
| PSMB1 | #620038 | *602017 | ?Neurodevelopmental disorder with microcephaly, hypotonia, and absent language |