Complex genomic rearrangement with deletion of PITX2 in a Chinese family with Axenfeld–Rieger syndrome: A case report and literature review

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: This study identified the genetic causes of Axenfeld–Rieger syndrome (ARS) in a Chinese family and evaluated their clinical phenotype and clinical treatment.

Methods: We recruited a Chinese family with ARS. The proband presented with bilateral ectopic pupils, periumbilical redundancy, craniofacial abnormalities, and dental abnormalities after birth and was diagnosed with ARS. The symptoms were the same for her younger brother. Blood samples were collected from four family members: the proband, her brother, and her parents. Whole-genome sequencing (WGS) was performed to identify probable genetic variants in the proband. To confirm the identified variants, samples from the other family members were subjected to quantitative polymerase chain reaction (qPCR) and Sanger sequencing.

Results: Based on the results of WGS, we suspected a deletion region and an inversion region around the PITX2 gene. Through qPCR and Sanger sequencing, we identified a complex rearrangement involving a 6.15 Mb deletion on Chromosome 4, including the PITX2 coding region (Hg38; chr4:110617776–116769011), a 45.71 Mb inversion (Hg38; chr4:116769011–162481408), and a 14-bp deletion (Hg38; chr4:162481409–162481422). Interestingly, the father’s copy number was normal, but Sanger sequencing revealed the same breakpoints. This indicated that the father is a balanced rearrangement carrier, and the children are unbalanced rearrangement carriers. While similar deletions and many breakpoints in this region have been reported, this specific rearrangement is novel.

Conclusions: Using WGS, qPCR, and Sanger, we found a complex genomic rearrangement with the deletion of PITX2 in a Chinese family with ARS. The clinical characteristics of the affected individuals were reported. The current findings broaden our understanding of the phenotype and variant spectrum associated with ARS caused by PITX2 deletion.

Axenfeld–Rieger syndrome (ARS; OMIM 180500, OMIM 601499, OMIM 602482) is an uncommon autosomal dominant disorder with an incidence of approximately 1 in 50,000 to 100,000 newborns [1]. The typical clinical manifestations of ARS include ocular and systemic phenotypes, and the disease is divided into three types. In ARS Type 1, eye involvement is typically bilateral or rarely unilateral. Bilateral iris hypoplasia, polycoria, iridocorneal adhesion, corectopia, posterior embryotoxon caused by the anterior displacement of Schwalbe’s line, and glaucoma are common ocular abnormalities [2]. Systemic manifestations include periumbilical redundancy and umbilical hernia [3–5]. Characteristic craniofacial features include maxillary hypoplasia, hypertelorism, telecanthus, a flattened midface with a broad, flat nasal bridge, a thin upper lip, and a prominent lower lip. Dental characteristics include microdontia, short roots, taurodontism, teeth with unusual shapes, and hypodontia/oligodontia of the primary and permanent dentition [6–8]. It is caused by variants in paired-like homeodomain transcription factor 2 (PITX2). At present, no single gene variant associated with ARS Type 2 has been found [9]. ARS Type 3 manifests as a variety of phenotypes, including eye defects, hearing loss, heart abnormalities, dental anomalies, and facial deformities. It is caused by variants in forkhead box C1 (FOXC1). The dental and facial malformations associated with Type 3 are distinct from those associated with Type 1. Facial deformities mostly include hypertelorism and ear anomalies, while tooth deformities mainly include enamel hypoplasia [1,5,6,9,10]. ARS has also been linked to two additional genes (CYP1B1, 2p22.2, OMIM 601771 [11] and PRDM5, 4q27, OMIM 614161 [12]) and one locus (13q14) [13].

Two key genes in ARS, FOXC1 (6p25, OMIM 601090) and PITX2 (4q25, OMIM 601542), have been identified using conventional genetic approaches. ARS Type 1 is caused by heterozygous variants of PITX2, while Type 3 is caused by heterozygous variants of FOXC1. Patients with ARS have a variety of PITX2 and FOXC1 variants, including point variants, insertion variants, deletion variants, and chromosomal deletions [1,14]. PITX2 encodes a bicoid homeodomain protein belonging to the RIEG/PITX homeobox family. It is involved in the development of the eyes, teeth, and abdominal organs [1]. ARS, iridogoniodysgenesis syndrome, and Peters anomaly are all linked to variants in this gene [15]. In this report, we present a novel complex genomic rearrangement with the deletion of PITX2 identified in two Chinese siblings with both ocular and systemic anomalies.

A large deletion spanning PITX2 and LOC107986306 (chr4:110617776–116769011) was found in the proband using WGS. The deletion is 6.15 Mb and contains 86 genes (Appendix 1), affecting cytogenic bands 4q25q26. The results of the variant analysis are shown in Appendix 2, with no pathogenic/likely pathogenic variants found in other genes. To verify the deletion and determine the exact DNA breakpoint, qPCR analysis and Sanger sequencing were performed for the four family members. The copy numbers of five predetermined candidate regions were quantified via qPCR. These were located in exon 4 of PITX2, exon 12 of ENPEP, exon 2 of FAM241A, exon 4 of NDST4, and exon 2 of NDST4. Compared to normal negative controls and their parents, the siblings’ expression levels of PITX2-e4, FAM241A, NDST4-e4, and NDST4-e2 suggested copy number deletions. There were no significant abnormalities in the copy number of ENPEP-e12 in both siblings. Neither parent had copy number deletions. In the experiment, we used two normal negative control genes, and the results were consistent (Table 1). Sanger sequencing revealed that the siblings and their father have two breakpoints on Chromosome 4. On Chromosome 4, the breakpoint 110617776 sequence is followed by 162481408, and the breakpoint 116769011 sequence is followed by 162481423. The sequences 162481409 to 162481422 have a small 14-bp deletion (Figure 3B). Thus, we identified a complex rearrangement involving a 6.15 Mb deletion on Chromosome 4, including the PITX2 coding region (Hg38; chr4:110617776–116769011) and a 45.71 Mb inversion (Hg38; chr4:116769011–162481408), and a 14-bp deletion (Hg38; chr4:162481409–162481422). However, Sanger sequencing showed that the breakpoints on Chromosome 4 are also present in the patients’ father. The translocation breakpoint removing a portion of the PITX2 3′UTR in the father did not have any clinical effects. We speculate that there is insertion translocation in the father, which is a complex chromosomal rearrangement that requires at least three breakpoints on the related chromosomes, and thus, he had no clinical symptoms. Insertion translocations are rare, balanced chromosomal rearrangements with an increased risk of imbalances for offspring. This suggests that the father is a balanced rearrangement carrier, while the children have unbalanced rearrangements. Additional analysis to identify the location of the insertion could not be performed due to limited sample availability. Combined with previous studies, we found that deletion affecting cytogenic bands 4q25q26 can cause different clinical manifestations (Table 3).

ARS is a group of clinically and genetically heterogeneous developmental disorders, which cause eye defects and non-ocular systemic defects. Ocular symptoms include anterior segment dysplasia of the eye with iris dysplasia, multiple pupils, posterior embryotoxon, and glaucoma [1,17]. ARS can also lead to non-ocular systemic defects, including distinctive craniofacial dysmorphism, hearing loss, dental abnormalities, umbilical skin defects, and heart defects [18,19]. ARS Type 1 usually features an ocular and systemic phenotype and is caused by variants in PITX2. In our study, WGS revealed that the entire coding region of the PITX2 genes was deleted in the proband. Based on their clinical presentation and genetic testing results, the patients in this study were classified as Type 1. Both had eye defects, dental abnormalities, craniofacial malformations, and umbilical abnormalities. On Chromosome 4, microdeletions near the PITX2 may also be associated with abnormal brain development [20 –23]. Intellectual disabilities were observed in the younger brother, with manifestations of neurodevelopmental abnormalities also present in the proband. The most dangerous complication of ARS is glaucoma, which develops in more than 50% of patients and can lead to total and irreversible blindness within a few years [24,25]. The proband was diagnosed with glaucoma.

On Chromosomes 4q25 and 6p25, PITX2 and FOXC1 variants account for 71% of the instances of ARS [9]. In addition to the typical intragenic variants (missense, nonsense, splicing, and intragenic deletion/insertion), large deletions affecting PITX2 and FOXC1 have been reported. PITX2 and FOXC1 encode developmentally relevant transcription factors that regulate the expression of downstream target genes by binding to specific DNA sequences. Transcription factors play an important role in embryonic development, and their expression is subject to strict spatiotemporal regulation. Therefore, variations in FOXC1 or PITX2 expression may lead to ARS [19,26–28]. PITX2 and FOXC1 act synergistically during individuals’ eye development, particularly that of the anterior segment. These two genes interact in the regulation of common downstream target genes in specific cell lines [29]. Interestingly, the variants in our case included not only two deletions but also an inversion. Although similar deletions and many breakpoints in the region harboring PITX2 have been reported, this specific rearrangement had not been reported. A description of ARS-associated deletions, which also include 4q25q26, as well as descriptions of their clinical manifestations, are provided in Table 3.

Herein, we identified two deletions and one inversion variant on Chromosome 4 in a family with ARS. Some of the clinical presentations in our patients were unusual and atypical for ARS. Among the 86 missing genes, we sought to identify genes associated with the patients’ symptoms. NEUROG2, UGT8, NDST4, and ZGRF1 were associated with cognitive impairment and neurodevelopmental phenotypes. Mental retardation was found in ARS patients harboring a 4q25q26 deletion [30], which includes NEUROG2 and UGT8. In another study, developmental delay was also reported in ARS patients with a 4q25-q28.2 deletion, including NEUROG2, UGT8, and NDST4 [19]. Similarly, Titheradge et al. reported ARS patients with 4q25q26 or 4q25-q27 deletions who had developmental delays, learning difficulties, difficulty with pronunciation, and autism spectrum disorder. In these cases, deleted genes included NEUROG2, UGT8, and ZGRF1 [21]. NEUROG2 is a transcription factor that belongs to the basic helix–loop–helix (bHLH) family and is involved in cell fate and neuronal differentiation within various areas of the central nervous system [31]. UGT8 is an enzyme of the uridine diphosphate glycosyltransferase family associated with the production of 3-O-sulfogalactosylceramide, a major component of the myelin sheath in the central and peripheral nervous systems [32]. NDST4 also plays an important role in the adult brain and embryonic development [33]. In a family chain analysis, ZGRF1 was found to be associated with childhood apraxia of speech, a severe form of speech sound disorder [34].

Despite extensive research, the potential genetic causes remain unknown in approximately 30% of ARS cases, with efforts in this direction still ongoing [9]. The current treatment for patients with ARS primarily focuses on glaucoma management. It is critical to regularly evaluate IOP and optic nerve development in patients owing the high risk of glaucoma and the fact that it is frequently diagnosed in childhood. Current glaucoma treatments include medications and surgery. However, reports have demonstrated that medical treatments do not successfully reduce IOP or hinder glaucoma progression in ARS patients with PITX2 or FOXC1 variants [35]. Surgical treatment has thus emerged as an alternative option, including laser iridotomy, laser trabeculoplasty, laser cycloablation, trabeculectomy, aqueous shunt devices, and minimally invasive glaucoma surgeries. The proband underwent micropulsed ciliary photocoagulation twice. Fortunately, the proband’s IOPs were temporarily controlled through surgery. In conclusion, our findings broaden the range of known PITX2 variants and may be useful for genotype–phenotype analyses in patients with ARS.

Appendix 1. Supplementary Table 1.

To access the data, click or select the words “Appendix 1.” Genes contained in the 4q25 and 4q26 deletion fragments(NCBI RefSeq Annotation GCF_000001405.40-RS_2023_03).

Appendix 2. Supplementary Table 2.

To access the data, click or select the words “Appendix 2.” Variants identified in the proband from WGS datasets.