Characterization of Associated Nonclassical Phenotypes in Patients with Deletion in the WAGR Region Identified by Chromosomal Microarray: New Insights and Literature Review

Abstract

WAGR syndrome (Wilms' tumor, aniridia, genitourinary changes, and intellectual disability) is a contiguous gene deletion syndrome characterized by the joint deletion of PAX6 and WT1 genes, located in the short arm of chromosome 11. However, most deletions include other genes, leading to multiple associated phenotypes. Therefore, understanding how genes deleted together can contribute to other clinical phenotypes is still considered a challenge. In order to establish genotype-phenotype correlation in patients with interstitial deletions of the short arm of chromosome 11, we selected 17 patients with deletions identified by chromosomal microarray analysis: 4 new subjects and 13 subjects previously described in the literature with detailed clinical data. Through the analysis of deleted regions and the phenotypic changes, it was possible to suggest the contribution of specific genes to several nonclassical phenotypes, contributing to the accuracy of clinical characterization of the syndrome and emphasizing the broad phenotypic spectrum found in the patients. This study reports the first patient with a PAX6 partial deletion who does not present any eye anomaly thus opening a new set of questions about the functional activity of PAX6.

Introduction

Interstitial deletions in the short arm of chromosome 11, which encompass the 11p13 region, are known to cause WAGR syndrome (MIM #194072). The syndrome is characterized by the presence of Wilms' tumor, aniridia, genitourinary alterations, and intellectual disability (mental retardation) [Yamamoto et al., 2013]. It is known that about 30% of the cases are diagnosed in childhood after aniridia identification [Tezcan et al., 2015].

Clinical diagnosis is confirmed by the detection of PAX6 (paired box 6) and WT1 (Wilms' tumor suppressor) gene deletions, therefore defining the syndrome as a contiguous gene syndrome [Hingorani et al., 2012]. WT1 plays an essential role in the development of urogenital and central nervous systems and is involved in hematopoiesis [Yang et al., 2007]. The major function of PAX6 is in ocular development regulation during embryogenesis; however, it has also been shown to have a role in pancreas, central nervous system, and olfactory system development [Osumi et al., 2008].

Reduction of PAX6 gene expression is the main cause of aniridia, which can manifest as part of the syndrome or as an isolated trait. Aniridia (MIM #106210) is an ophthalmopathy characterized by the absence or incomplete formation of the iris and hypoplasia of the fovea, resulting in decreased visual acuity and nystagmus. This pathology is usually identified during childhood and may be associated with several other ocular alterations [Lee et al., 2008; Hingorani et al., 2012].

WT1 gene deletion is associated with an increased risk of Wilms' tumor (nephroblastoma), thus making renal monitoring of WAGR patients essential [Hingorani et al., 2012]. Reproductive system malformations may also occur as the WT1 gene participates in gonadal development [Hastie, 2017]. In addition, a high incidence of obesity and behavioral problems have been reported in WAGR patients [Shinawi et al., 2011].

Due to the significant variation of deletion size (0.6–23 Mb), a small critical region of 0.6 Mb encompassing the PAX6, RCN1, and WT1 genes defines the syndrome [Xu et al., 2008]. More complex, atypical WAGR phenotypes are associated with deletions that extend to the telomeric and/or centromeric regions thereby affecting several other genes.

In order to establish genotype-phenotype correlations in patients with interstitial deletions in the short arm of chromosome 11 encompassing PAX6 and WT1 and larger than the established minimal region, we present 4 new subjects. In addition, we present a comparison of both the clinical and molecular characteristics of these patients with selected cases described in the literature. Collective analysis of these deletions enabled us to propose the contribution of specific genes in other clinical manifestations observed in WAGR syndrome.

Materials and Methods

Patient Selection

Four patients were selected at the Medical Genetics Service of the University Hospital of Brasília. Three patients were included in the study due to the presence of syndromic forms of aniridia. One patient without aniridia had a 11p13 deletion identified through standard diagnostic analysis for multiple congenital malformations.

Karyotype

The G-banding technique was performed according to standard procedures.

Chromosomal Microarray Analysis

Chromosomal microarray analysis (CMA) was performed using the CytoScan^TM 750K platform (ThermoFisher®, Carlsbad, USA). CytoScan^TM 750K chips feature approximately 750,000 probes that allow high-resolution analysis of copy number variations and single nucleotide polymorphisms. DNA hybridization to the chip was performed according to the manufacturer's instructions and was analyzed using the GeneChip® Scanner 3000 7G (ThermoFisher). The data were analyzed using the Chromosome Analysis Suite (ChAS) software (ThermoFisher).

Literature Review

The studies selected in this review were sourced from the main public databases, primarily PubMed and Medline. The keywords searched were: WAGR syndrome, aniridia, 11p13 deletion, WT1 deletion, PAX6 deletion, aCGH and microarray, both in isolation and combined. Only subjects with PAX6 and/or WT1 deletions identified by CMA (oligoarray, aCGH or SNP-array) were selected and included in this review. In addition, studies that did not present coordinates and/or a complete clinical description were excluded. The patients reported in this study (P1–P4) were analyzed in conjunction with the selected cases from the literature (P5–P17), detailed in Tables 1 and 2.

Table 1.

Reported cytogenetic findings

Patient	Reference	Deletion size, Mb	Deleted genes, n	Coordinates	Method	Karyotype
P1	Present study	10	41	Chr11:24,315,244–34,478,570	CMA	46,XX
P2	Present study	1.8	8	Chr11:30,477,356–32,240,306	CMA	46,XX
P3	Present study	19.2	65	Chr11:20,640,710–39,867,624	CMA	*
P4	Present study	4.9	38	Chr11:31,813,872–36,773,780	CMA	46,XX
P5	Hu et al., 2015	0.518	3	Chr11:31,486,211–32,004,859	SNP-array	*
P6	Hu et al., 2015	0.518	3	Chr11:31,486,211–32,004,859	SNP-array	*
P7	Yamamoto et al., 2013	8.6	45	Chr11:29,676,434–38,237,948	aCGH	*
P8	Huynh et al., 2017	5.8 (in mosaic)	38	Chr11:31,521,664–36,801,306	aCGH	mos 46,XY,del(11)(p13)[9]/46,XY[11]
P9	Takada et al., 2017	14.5	56	Chr 11:24,792,569–39,222,929	aCGH	46,XX,del(11)(p11.2p14)
P10	Seo et al., 2018	14.5	68	Chr11:22,004,830–36,501,579	aCGH	46,XY,inv(3)(q13.2q21),del(11)(p11.2p13)
P11	Tezcan et al., 2015	7.2	41	Chr11:30,863,700–38,018,632	aCGH	*
P12	Rodríguez-López et al., 2013	17.8	75	Chr11:18,676,926–36,576,388	aCGH	46,XX,del(11)(p15.1p12)
P13	Dolan et al., 2011	5.5–5.8	33	Chr11:29,404,793–35,232,710	aCGH	46,XY,del(11)(p13p14)
P14	Gimelli et al., 2010	˜14.6	55	Chr11:25,424,279–40,016,396	aCGH	*
P15	Meng et al., 2020	26.25	89	Chr11:18,742,043–44,991,839	SNP-array	*
P16	Le Caignec et al., 2007	˜10	45	Chr11:29,414,215–38,667,898	aCGH	46,XY,del(11)(p12p14.1)
P17	Sheehan et al., 2009	9.5	46	Chr11:27,956,661–37,509,625	aCGH	46,XY

Coordinates converted to Human Genome version 19.

^*Not available..

Table 2.

Patient age and clinical findings

Patient	Age	A	GA	WT	ID	SS	ND	CD	P/C	RD	NSD	Other ocular findings	Other findings/description
P1	4 years	+	−	−	+	+	−	+	+	−	−	−	Umbilical hernia
P2	9 years	+	−	−	+	−	−	−	−	−	−	−	Bilateral epicanthus
P3	2 years	+	+						+	+	+	Nystagmus	Horseshoe kidney in iliac position, accessory spleen, resistance to finger extension
P4	6 years		+	+	+			+		+	+		Microcephaly, brachycephaly, small and low-set ears, midface hypoplasia, posterior cleft palate with bifid uvula, umbilical hernia, hyperconvex nails, hypoplastic labia minora, leukomalacia, corpus callosum thinning, polycystic kidneys
P5	31 years	+										Ptosis, cataract	−
P6	26 years	+	−	−	+	−	−	−	−	−	−	−	Prominent forehead, short philtrum, intellectual disability
P7	3 years	+			+	+	+		+			Ptosis, cataract, glaucoma, corneal opacity, strabismus, staphyloma (right eye)	Autistic behavior, depressed nasal bridge, downslanting palpebral fissures, low-set ears
P8	2 years	+	−	+	*							Congenital hypothyroidism, ectopic thyroid, presence of left retroperitoneal mass in the neonatal period
P9	2 years	+	−	+	+	−	+	−	−	−	−	Cataract, macular hypoplasia	Absence of speech
P10	4 years	+	+	+	+	−	+	+	−	+	−	Cataract, macular hypoplasia	Tiny cyst (left kidney), inversion at chromosome 3
P11	−	*	*	*	*	*	*	+	*	*	+	−	Absence of the corpus callosum, cavum septum pellucidum and intrauterine growth retardation
P12	3 years	+											Obesity
P13	Newborn	+	*	*	*	*	*	+	*	*	*	Cataract	Large volume of ascites, bilateral communicating hydroceles
P14	7 months	+										Glaucoma	Short philtrum, thin upper lip, bilateral mild overlapping of the 2nd and 4th toes over the 3rd
P15	6 months	+	−	−	+	−	+	+	−	−	−	Ptosis, nystagmus, macular dysplasia	Enlarged anterior fontanel, low-set ears, micrognathia
P16	5 months	+						+	−	+	+	Cataract, nystagmus	Low-set right malformed ear, segmental glomerulosclerosis, supernumerary spleen, ovaries not observed
P17	3 months	+	−	−	−	−	−	+	−	−	−	−	Erythema with diffuse peeling of skin, seborrheic dermatitis, hair paucity

A, aniridia; GA, genitourinary alterations; WT, Wilms’ tumor; ID, intellectual disability; SS, short stature; ND, neurodevelopmental disorders; CD, cardiac disorders; P/C polydactyly/clinodactyly; RD, renal disorders; NSD, nervous system disorders.

^*Not available.

Results

Patient 1

A 4-year-old girl, the daughter of nonconsanguineous parents, was referred to the Genetics Service due to developmental delay. On physical examination, she presented horizontal nystagmus, depressed nasal bridge, underdeveloped alae nasi, a short and marked philtrum, a wide mouth, thick lips, retrognathia, and prominent ears. The patient had an umbilical hernia, deep palmar creases, bilateral third finger clinodactyly, overlap of the second and third toes, normal female external genitalia, and short stature (<3rd centile). Investigations revealed the presence of a patent foramen ovale and pulmonary stenosis. The ophthalmologic evaluation detected transparent cornea, total bilateral aniridia, cataracts, and bilateral horizontal pendular nystagmus.

Karyotype analysis was normal (46,XX). CMA revealed a 10-Mb interstitial deletion in the short arm of chromosome 11 involving bands 11p13p14.3 (arr[hg19] 11p13p14.3(24,315,244_34,478,570)×1). The deletion encompasses 41 genes, including PAX6 and WT1.

Patient 2

A 9-year-old female patient, the daughter of nonconsanguineous parents, was referred to the Genetics Service due to a clinical diagnosis of aniridia. Physical examination revealed bilateral aniridia, bilateral epicanthal folds, arched eyebrows, a narrow forehead, depressed nasal bridge, a short nose, small mouth, high and narrow palate, low-set ears, obesity (97th centile), and intellectual disability.

The karyotype did not reveal alterations (46,XX). CMA detected a 1.8-Mb deletion in the 11p14.1p13 region (arr[hg19] 11p14.1p13(30,477,356_32,240,306)×1). The deletion encompasses PAX6 and a further 7 genes, not including the WT1 gene.

Patient 3

A 2-year-old male patient, the second child of a nonconsanguineous young couple, was delivered by cesarean section at 38 weeks of gestation and presented low birth weight (2,375 g, <3rd centile). Ophthalmologic evaluation detected bilateral partial aniridia. Abdominal ultrasonography showed a horseshoe kidney in an iliac position and a small accessory spleen.

Physical examination revealed a wide anterior fontanelle, narrow palpebral fissures, epicanthal folds, vertical nystagmus, anteverted nares, a long philtrum, high palate, retrognathia, overlapping of the third and fourth toe on the right foot, and overlapping of the second and third toe on the left foot, together with clinodactyly of the fourth right toe. The patient presented a normal penis, right cryptorchidism, and a muscular tone suitable for his age, but with resistance for finger extension.

CMA identified a 19.2-Mb deletion in the short arm of chromosome 11 involving bands 11p12p15.1 (arr[hg19] 11p12p15.1(20,640,710_39,867,624)×1). The deletion encompasses 65 genes including PAX6 and WT1. Karyotype analysis was not performed.

Patient 4

A 6-year-old female patient, the second child of nonconsanguineous parents, was referred to the Genetics Service at 19 months of age. The patient was born by vaginal birth, preterm at 30 weeks and 6 days of gestation, with a birth weight of 1,380 g (10th–50th centile), length of 36 cm (<3rd centile), and head circumference of 25 cm (<3rd centile). Due to prematurity, she spent 30 days in the Intensive Care Unit. At 19 months she was diagnosed with Wilms' tumor, which was surgically removed, followed by chemotherapy cycles.

Physical examination revealed microcephaly (OFC <3rd centile), brachycephaly, ocular hypertelorism, small and low-set ears, midface hypoplasia, posterior cleft palate with bifid uvula, umbilical hernia, hyperconvex nails, hypoplastic labia minora. The patient also had significantly delayed neuropsychomotor development and intellectual disability and needed nasogastric tube feeding. Complementary examinations revealed patent ductus arteriosus, patent foramen ovale, pericardial effusion, periventricular leukomalacia, corpus callosum thinning, ventricular system dilation, and polycystic kidneys. Ophthalmologic examination did not reveal aniridia or other eye defects.

Karyotype analysis did not disclose any abnormalities (46,XX). CMA identified a 4.9-Mb deletion in the short arm of chromosome 11 affecting the bands 11p13p12 (arr[hg19] 11p13p12(31,813,872_36,773,780)×1). The deletion fully encompasses the WT1 gene, together with 36 other genes, and part of the PAX6 gene.

Discussion

Though WAGR syndrome is characterized by joint loss of PAX6 and WT1, many patients do not have all the classical alterations and have deletions that encompass other genes, making the phenotype of the syndrome highly variable [Hingorani et al., 2012].

The use of classical and molecular cytogenetic techniques is important to confirm the diagnosis of WAGR syndrome. However, although karyotyping has been considered the gold standard technique for detecting chromosomal alterations for many years, it has limitations regarding the size of the chromosomal rearrangement that can be observed [Cheung and Bi, 2018]. In this way, some changes may go unnoticed by the conventional cytogenetic technique, as it has a limited detection range and may not detect rearrangements below 5–10 Mb [Miller et al., 2010].

In recent years, a more robust and high-resolution tool has become available. CMA allows more precise identification of breakpoints, providing high sensitivity and specificity to the technique [Blanco-Kelly et al., 2017]. In our cohort only P3 had a 19.2-Mb deletion that would undoubtedly be identified in the karyotype. P1 had a 10-Mb deletion that was not noticed in the karyotype probably for being exactly at the resolution range.

Genotype-Phenotype Association

Approximately 10% of aniridia cases are caused by chromosomal rearrangements involving the PAX6 gene, either partially or entirely. In the case of large deletions, adjacent neighboring genes are deleted together, such as the WT1 gene leading to WAGR syndrome, constituting a contiguous gene disorder [Compton et al., 1988]. Although the phenotype associated with WAGR syndrome is well-characterized, large clinical variability can be observed due to nonrecurrent breakpoints, and therefore, deletions that include different genes along with PAX6 and WT1 can result in complex phenotypes [Dolan et al., 2011].

According to Shinawi et al. [2010], genotype-phenotype correlation can be performed by construction of an exclusion map, comparing the phenotypic changes of patients with different portions of 11p deletions. Figure 1 shows the different deletion sizes of the patients analyzed in this study.

Fig. 1.

Schematic representation of deleted regions in the subjects analyzed. Horizontal bars represent the deleted segments. Literature review cases are represented by light gray bars, the cases analyzed in this study are represented by dark gray bars. All genes deleted in the analyzed region are shown below the idiogram. Some genes that contribute to phenotypic manifestations are depicted above.

Fischbach et al. [2005] identified the main classical and nonclassical findings of WAGR syndrome in a review of 54 cases of clinically diagnosed patients without molecular testing. Classical findings include Wilms' tumor, aniridia, intellectual disability, and genitourinary alterations. Nonclassical clinical signs include facial dysmorphisms and neurological, cardiopulmonary, renal, musculoskeletal, metabolic, and gastric defects.

There is no consensus on the criteria for defining the clinical diagnosis of WAGR syndrome. Although the syndrome is characterized by 4 classical clinical signs, it is not mandatory to present all of them. Aniridia is the main manifestation, being generally diagnosed at birth. Alterations in the external genitalia are identified mainly in male individuals, since they are not present in most affected girls; and Wilms' tumor is reported in about 45–57% of WAGR cases [Clericuzio, 2010].

Therefore, the clinical diagnosis can only be confirmed through molecular evaluation. Children with aniridia or some other classical manifestation should undergo a chromosomal study in search of deletions in the 11p13 region [Clericuzio, 2010]. According to Fischbach et al. [2005], the diagnosis of aniridia in young children is enough to initiate the search for WAGR syndrome. In older children, the authors recommend the presence of aniridia and another classical clinical sign to begin the investigation. The recommendations for WAGR syndrome management can be seen in Fischbach et al. [2005]; the authors provide a guideline for health supervision with recommendations for medical treatment, education, and integration into the family and society.

According to these criteria, all new cases presented in this study had an indication of clinical diagnosis of WAGR syndrome.

In agreement with literature reports, we observed clinical features such as short stature, heart defects, renal disorders, accessory spleen, central nervous system, and other eye defects in our patients. In addition to these findings, neurodevelopmental disorders and polydactyly were also identified in selected patients in the literature.

Wilms' Tumor

In 4 subjects (P4, P8, P9, and P10) Wilms' tumor was reported, a classical WAGR syndrome manifestation. Our data corroborate literature reports that show that only around 30% of WAGR patients are at risk of developing Wilms' tumor during childhood [Dumoucel et al., 2014].

According to Millar et al. [2017], Wilms' tumor is a type of nephroblastoma that mainly affects children under 5 years of age. The manifestation can be unilateral or bilateral, the latter form usually occurs at an earlier age. Tumor management is essential to achieve remission with as little morbidity as possible, starting with an early diagnosis. Surgical excision, the use of chemotherapy and radiotherapy, and in some cases the need for kidney transplantation, should be evaluated.

The study published by Wang et al. [2019] provides a review of diagnostic and treatment strategies, highlighting the main similarities and differences between the National Wilms Tumor Study Group (NWTSG)/Children's Oncology Group (COG) and The International Society of Paediatric Oncology (SIOP), the 2 main guidelines used in Wilms' tumor management today.

The WT1 gene plays an essential role in kidney development and can function as a tumor suppressor or an oncogene. For instance, WT1 variants can result in a gain of function and lead to exacerbated cell proliferation. Studies in kidney tumor cells identified a role of WT1 in cell proliferation possibly due to its interaction and antagonistic action with the p53 protein [Tian et al., 2014]. p53 has a tumor suppressor function, and the loss of its function has already been detected in different types of cancer [Muller and Vousden, 2014].

Variants affecting the DNA-binding domain result in loss of function, as is the case when the WT1 gene is deleted [Al-Hussain et al., 2014]. Inactivation of WT1 can lead to cell survival and proliferation, thus contributing to tumor development. This balance between oncogenic and tumor suppressive action is not yet fully understood. However, it is known that the determination of this function is the result of specific cellular conditions during development and how the organism responds to changes in WT1 expression [Toska and Roberts, 2014].

In addition, a study by Marakhonov et al. [2019] found that the simultaneous deletion of WT1 and LMO2 increases the risk of developing a kidney tumor to 90%. The LMO2 gene functions as a tumor suppressor, and its expression is reduced in different types of solid cancers.

Aniridia

Aniridia was the clinical manifestation observed in all subjects analyzed, except for P4. Only 2 patients have isolated aniridia (P5 and P6), which can be explained by the small deleted segment in these patients, comprising only the IMMP1L, ELP4, and PAX6 genes. In 14 subjects, aniridia is related to WAGR syndrome while in 3 of these subjects WAGR is also associated with Peters' anomaly (P13), Ommen syndrome (P17), or Potocki-Shaffer syndrome (P15).

According to Kokotas and Petersen [2010], approximately 10–13% of aniridia cases are related to WAGR syndrome, whereas 85% of cases are either the result of single nucleotide variants or altered expression of the PAX6 gene only. The remaining 2–3% of the cases are related to other syndromes. Our data corroborate a study by Wawrocka et al. [2013] who analyzed 12 patients with aniridia, 5 of which had a deletion in 11p13, 4 had a variant in the PAX6 gene, while 1 had an intact PAX6 gene, indicating a position effect due to loss of downstream regulatory elements as the probable cause of aniridia.

Davis et al. [2008] described an individual with aniridia, autism, and moderate intellectual disability. The patient presented a 1.3-Mb deletion, including the MPPED2, DCDC5, DPH14, IMMP1L, and ELP4 genes, in addition to the PAX6 regulatory element. This case shows that a deletion close to PAX6 and its regulatory element can interfere with gene expression and generate a similar phenotype to the gene deletion. Ansari et al. [2016] analyzed 13 patients with aniridia, 9 of which were due to the PAX6 deletion, 3 presented alterations in FOXC1 gene expression, 2 had de novo single nucleotide variants, and 1 presented a telomeric deletion of the PITX2 gene.

Although PAX6 deletions play an important role in the etiology of aniridia, there are several molecular causes of aniridia that should be highlighted. Aniridia can hence be caused by a single nucleotide variant, total or partial deletion of PAX6, by a position effect such as variants/deletions that encompass regulatory elements that would affect PAX6 gene expression, but also by altering the expression of other genes that influence eye development, such as FOXC1, PITX2, and PITX3 [Hingorani et al., 2012].

Lima Cunha et al. [2019] also showed that in addition to partial or complete iris hypoplasia, other eye abnormalities are observed in patients with aniridia. Of the 17 subjects analyzed, 10 presented other ocular findings associated with aniridia, such as cataract, nystagmus, ptosis, glaucoma, hypoplasia and macular dysplasia, corneal opacity, strabismus, and staphyloma. These findings corroborate the observations of Fischbach et al. [2005] in a study involving 54 subjects, in which 36 presented cataracts, 24 glaucoma, and 22 nystagmus. The presence of optic nerve hypoplasia, macular/foveal hypoplasia, retinal detachment, strabismus, ptosis, and corneal pannus was also observed in a smaller number.

PAX6 Partial Deletion without Eye Malformation

A partial deletion of the PAX6 gene was identified in patient P4. Despite being diagnosed with WAGR syndrome due to a 4.9-Mb deletion in 11p (Fig. 2), the patient does not have aniridia or any other ocular manifestation described in previous PAX6 deletion cases (Fig. 3).

Fig. 2.

Chromosomal microarray analysis of patient P4. The red bar represents the deleted region. The break point, indicated by the dotted vertical line, shows the partial deletion of the PAX6 gene. The red arrows indicate the preserved exons.

Fig. 3.

Patient P4 at the age of 19 months and 8 years, presenting several facial dysmorphisms, ocular hypertelorism, depressed nasal bridge, anteverted nares, and small teeths.

Bobilev et al. [2016] described 2 individuals diagnosed with aniridia and a partial PAX6 deletion. In 1 patient, the deletion extended from the P1 promoter to exon 4, while in the other patient exons 6 and 7 were deleted. According to the authors, the partial deletion resulted in reduced PAX6 function in both subjects. Bozkaya et al. [2016] described a family case of aniridia associated with congenital cataracts due to exon 8 deletion.

Ramirez-Miranda and Zenteno [2006] reported intragenic deletion of PAX6 in 4 unrelated subjects of isolated aniridia. They identified deletions in exons 6 and 7, which led to the formation of a premature termination codon, and in exon 9. An exon 9 deletion was also described by Redeker et al. [2008], in addition to 2 subjects in which a deletion was detected that extended from exon 8 to 14 in aniridia patients. In our study, CMA showed that only PAX6 exons 12 and 13 are preserved in patient P4, unlike any other report in the literature.

The PAX6 protein has 2 DNA-binding domains (PD and HD) and a transactivation domain, which are encoded by exons 4–7, 8–10, and 10–13, respectively. The transactivation domain plays a role in recruiting different co-activators for enhancers through protein-protein interaction, the 3 exons that encode it act synergistically to achieve the full level of transcription activation [Shaham et al., 2012]. Therefore, the transactivation domain is essential for PAX6 function as a transcription factor. This C-terminal domain is highly conserved and found in all genes in the PAX family [Monsoro-Burq, 2015].

In addition to acting in isolation, the PAX6 gene can be associated with other transcription regulators at specific PAX6 recognition sites [Shaham et al., 2012]. Kamachi et al. [2001] identified that PAX6 can act synergistically with SOX2 in a chicken embryo in early lens development. Both transcription factors can recognize the same site, which differs from the optimal PAX6 recognition sequence, P6CON.

According to Cvekl et al. [2004], tissue-specific gene expression is coordinated by a combination of active transcription factors, and genes are expressed simultaneously in a given tissue. The PAX6 gene is expressed in all tissues involved in the development of the eye, however, it does not act alone. In lens development, for example, it acts in synergy with several transcription factors (c-Maf, MafA/L-Maf, MafB, NRL, Sox2, Sox1, RARß/RXRß, RORa, Prox1, Six3, FBP-B and HSF2), which are differentially expressed during the development stages. In addition, chromatin co-activators and remodelers regulate the accessibility and activity of PAX6 and other DNA-binding factors.

Thus, we propose a number of hypotheses that may account for why patient P4 has a partial deletion of PAX6 and does not present the expected phenotype.

Since PAX6 is able to act in synergy with a variety of transcription factors that recognize the same consensus sequence, it is possible that one or more of these proteins, in the absence of PAX6, could fully or partially fulfill a function primarily executed by PAX6 at a key point in eye development resulting in normal ocular development. Function redundancy is an important mechanism in maintaining organism integrity and is commonly found within a family of transcription factors that collaborate synergistically to allow specific function execution, essential to cell fate. An example of this process is considerable redundancy and functional compensation between SOXF family members, a subgroup of the SOX family transcription factors. The SOX7, SOX17, and SOX18 genes are required together in cardiac, vascular, and lymphatic development [Lilly et al., 2017].

Another possibility could be the expression of non-deleted exons. The rearrangement that occurred at the breakpoints may have favored the expression of exons 12 and 13. It is known that PAX6 has regulatory sequences conserved in the 3′ region and that there are more distant regulatory elements located 130 kb downstream from the poly-A tail [Shaham et al., 2012]. The expression of these exons, which encode the transactivation domain, could also contribute to the normality of ocular development. Since the PST domain operates in recruiting co-activators necessary for complete PAX6 activation, this activity could lead to the overexpression of the preserved allele maintaining the optimal level of PAX6 protein activity. Though this could be theoretically speculated, we were not able to perform any former tests in our patient or identify similar cases in the literature.

Mosaic 11p13 deletions have been reported in 5 cases and one of them was phenotypically normal [Robinson et al., 2008]. This patient has an affected monozygotic twin, reported by Crolla and van Heyningen [2002]. The authors suggest that the normal phenotype presented by one of the twins is due to the low frequency of the mosaicism and/or the differential distribution of the deletion in the cell lines of each one. From the CMA analysis, there is no evidence of mosaicism in patient 4, however confirmatory tests and analysis of other tissues could not be performed.

The role of regulatory elements within the deleted region or near the breakpoints should also not be disregarded.

Therefore, the presence of a partial deletion of PAX6 without the phenotypic manifestation expected suggests that there is a mechanism that compensates for this alteration. However, further functional studies should be conducted to determine what this mechanism is and how it interferes with ocular development, since to our knowledge this constitutes the first report of a PAX6 partial deletion without an eye anomaly phenotype.

Genitourinary Malformation

Genital system malformations were reported in 3 patients. P3 and P10 presented cryptorchidism, while P4 had hypoplastic labia minora.

Commonly, female individuals with WAGR syndrome have normal external genitalia, while male individuals may have gonadal dysgenesis, hypospadias, bifid scrotum, and cryptorchidism with possible genital ambiguity [Blaschko et al., 2012; Hutson et al., 2014].

A study by Lim et al. [2001] identified that the WT1 gene, together with androgen hormones, modulates testicular descent. Therefore, the deletion of WT1 can interfere in this modulation, thus contributing to the establishment of cryptorchidism.

Intellectual Disability and Neurodevelopmental Disorders

Intellectual disability/developmental delay was reported in 8 patients (P1, P2, P4, P6, P7, P9, P10, and P15), with some of them also presenting autistic behavior and speech delay.

The review conducted by Fischbach et al. [2005] identified 39 patients with intellectual disability/developmental delay in a total of 54 cases of WAGR syndrome, with a higher frequency in males. In addition to the PAX6 and WT1 genes that act in central nervous system development [Yang et al., 2007; Osumi et al., 2008], other genes present in the short arm of chromosome 11 can also contribute to the manifestation of these phenotypes.

A study by Xu et al. [2008] aimed to identify genes that contribute to intellectual disability and autism in patients with WAGR syndrome. The authors analyzed 31 patients with 11p deletion and observed that BDNF and SLC1A2 genes were deleted in most patients, while PRRG4 was deleted in all of the subjects analyzed.

BDNF is expressed in cortical neurons and plays an essential role in the survival of striated neurons. The SLC1A2 gene encodes a glutamate transporter, which in excess has neurotoxic effects. The PRRG4 gene encodes an integral membrane protein with an intracellular portion involved in signal transduction, cell cycle progression, and other cell functions. Therefore, the authors suggest that the haploinsufficiency of these genes contributes to intellectual disability and behavioral problems in patients with WAGR syndrome [Xu et al., 2008].

A study by Han [2016] aimed to determine whether BDNF deletion contributes to the manifestation of neurocognitive abnormalities in subjects with WAGR syndrome. In this evaluation of cognitive function in 24 subjects with WAGR syndrome (13 BDNF ^+/− and 11 BDNF ^+/+), the authors reported 10 subjects with isolated aniridia and 20 healthy controls, concluding that BDNF haploinsufficiency seems to be a modulating factor for more severe damage in adaptive behavior and cognitive functioning in subjects with 11p deletions.

Addis et al. [2015] identified a deletion of the ELP4 gene in 6 patients with a neurological phenotype, including speech delay, autism, developmental delay, and epilepsy. In addition, it was possible to observe that almost half of the 24 patients analyzed in the referred study presented difficulties in speech and language, supporting the hypothesis that the ELP4 locus influences language development.

The ELP4 gene is a subunit of the elongation factor of the acetyltransferase complex, associated with RNA polymerase II [Winkler et al., 2001] and is associated with central nervous system disorders, including intellectual disability and epilepsy [Chaverra et al., 2017]. Clinically, language development problems are related to the autistic spectrum [Eicher and Gruen, 2015]. Genetic studies have already reported evidence of linkage of several genomic regions with autism, including the short arm of chromosome 11 [Newbury et al., 2009].

Other Nonclassical Phenotypes Observed in 11p Deletions

Renal Disorders

Patients P3, P4, P10, and P16 presented kidney disorders. The molecular evaluation revealed extensive deletions involving multiple genes that are expressed, or have a role, in kidney development, namely: ELF5 which acts as a tumor suppressor and is highly expressed in the kidneys [Lapinskas et al., 2011]; CAT, that codifies the synthesis of the catalase enzyme and, according to Mohammedi et al. [2013], has a variant allele associated with diabetic nephropathy; BBOX1, which encodes an enzyme necessary for the synthesis of L-carnitine, essential for the oxidation of fatty acids, and is highly expressed in the kidneys [Rigault et al., 2006]; and SLC5A12, that also has high expression in the kidneys and encodes a transporter of glucose and other sugars [Barat et al., 2016].

Another relevant gene deleted in P3, P4, and P10 is LGR4; its homozygous inactivation in rats results in hypoplastic kidneys [Mohri et al., 2012]. A study published by Yi et al. [2014] demonstrated that Lgr4 inactivation in mice leads to multiple alterations found in WAGR syndrome, including small and polycystic kidneys. Therefore, they suggest that LGR4 is a potential candidate gene in the pathogenesis of WAGR syndrome.

Horseshoe kidney with an ectopic position was a clinical finding observed in patient P3. This abnormal kidney shape is the most commonly reported renal malformation and is characterized by fusion of the lower poles of the kidney, generating a horseshoe aspect [Sakala and Dyer, 2015]. There is no known genetic cause; however, the horseshoe kidney was reported in twin siblings and members of the same family [Natsis et al., 2014], suggesting a genetic cause. To our knowledge, the only other case of horseshoe kidney, with Wilms' tumor, in patients with WAGR syndrome was reported by Fantes et al. [1992].

On the other hand, despite not being a recurrent observation in patients with 11p deletion, polycystic kidneys were identified in patients P4 and P10, also diagnosed with Wilms' tumor. Gucev et al. [2011] reported a case of WAGR syndrome with bilateral polycystic kidney. The presence of multiple renal cysts can be attributed to several genes such as PAX2, PKD1, PKD2, and HNF1B [Eneman et al., 2014], none of which is mapped in the short arm of chromosome 11.

In addition to its role in renal development [Chau and Hastie, 2012], WT1 haploinsufficiency was reported as a possible cause of glomerulosclerosis [Lijima et al., 2012]. Therefore, the deletion of these genes may provide evidence of the genetic cause of the horseshoe kidney and polycystic kidneys in patients with the 11p13 deletion.

Short Stature

Short stature and growth deficit were reported in 3 patients (P1, P7, and P11). To our knowledge, there are no reports of any gene in the critical region for the WAGR syndrome that explains this clinical finding. However, Dateki et al. [2016] described a patient with a 9.2-Mb deletion in 11p14.1p15.3 with short stature and late closure of the fontanelles, probably explained by deletion of the NELL1 gene.

The NELL1 gene encodes a neural epidermal growth factor, similar to EGF, expressed mainly in the central nervous system [Nakamura et al., 2012]. Although not deleted in the patients with short stature analyzed in this study, the NELL1 gene is in close proximity to the deletions. It is possible that the expression of NELL1 may be influenced by position effect or by the loss of a regulatory element present in the deleted region.

However, one cannot exclude the influence of other factors that may also contribute to the establishment of short stature, such as nutritional status in the first 2 years of life, the influence of other genes in this phenotypic manifestation, and presence of other variants such as growth hormone deficiency, for example [Grunauer and Jorge, 2018].

Cardiac Disorders

Cardiac defects were observed in 8 of the 17 subjects analyzed: P1 − pulmonary stenosis and patent foramen ovale; P4 − patent ductus arteriosus, patent foramen ovale, and pericardial effusion; P10 and P15 − septum atrial defect; P11 − bilateral ventriculomegaly; P13 − atrial and ventricular enlargement; P16 − bicuspid aortic valve; and P17 − holosystolic murmur.

The review by Fischbach et al. [2005] identified the following manifestations in WAGR subjects: patent foramen ovale, pulmonary hypertension, valvular hypoplasia, ventricular septal defect, patent ductus arteriosis, tetralogy of Fallot, and atrial septal defect in 20.37%, a frequency lower than in our study.

The deletions in subjects with cardiac disorders analyzed in this study have a small overlapping region which involves the WT1, RCN1, and CD59 genes. The WT1 gene is expressed in the transverse septum influencing heart development in early development [Chau and Hastie, 2012]. RCN1 directly regulates calcineurin, a protein activated by Ca²⁺, whose elevated expression in the developing heart induces cardiac hypertrophy [Hilioti et al., 2004]. Finally, reduction of CD59 expression was described in patients with CHIME syndrome, characterized by coloboma, heart defects, intellectual disability, hearing abnormalities, among other changes [Ng et al., 2012].

In addition, other genes in the deleted region can also contribute to congenital cardiac defects present in these patients: PDHX, associated with non-ischemic dilated cardiomyopathy [Kao et al., 2015]; EHF, which has differential expression detected in acute myocardial infarction [Cao et al., 2017]; and RAG1, associated with cardiac fibrosis and iron overload in rats when underexpressed [Santos et al., 2000].

Therefore, even though there is no direct association, it is possible that these genes contribute to the cardiac alterations reported.

Polydactyly

Among the musculoskeletal abnormalities, polydactyly was observed in patients P1, P3, and P7.

One of the deleted genes in patients P1 and P3 was LGR4, that acts as a TNF receptor and negatively regulates osteoclast differentiation and bone resorption [Luo et al., 2016]. Wuyts et al. [2004] described 2 patients with 11p proximal deletion syndrome (P11pDS) who, among other alterations, presented small hands and feet.

A case of bilateral preaxial polydactyly associated with WAGR syndrome was described by Manoukian et al. [2005]. The authors proposed the ALX4 gene, located in the 11p11.2 region, as a possible candidate gene to explain polydactyly, since a variant in the orthologous gene was reported in mice with preaxial polydactyly in both anterior and posterior limbs.

ALX4 is a transcription factor expressed in mesenchymal condensations and has a role in the development of various organs and tissues, including bone and limb development [Shi et al., 2017]. However, the ALX4 gene is not mapped in the critical region for WAGR syndrome.

Although not recurrent, malformations of the feet and hands are found in patients who have a deletion in 11p, as described by Brémond-Gignac et al. [2005], who reported 3 cases of halucal polydactyly in patients with WAGR syndrome. There is no knowledge of genes present in 11p to date to explain these clinical findings, however the ALX4 and LGR4 genes may provide clues to the cause of these nonclassical clinical findings.

It is important, however, to remember that polydactyly can be caused by a variety of genetic events and that familiar polydactyly should also be discarded.

Central Nervous System Disorders

Malformations in the central nervous system were observed in patients P4, who presented microcephaly with partial agenesis of the corpus callosum and periventricular cystic leukomalacia, and P11, with complete agenesis of the corpus callosum and septum pellucidum.

Agenesis of the corpus callosum is the most common brain malformation described in patients with WAGR syndrome. Though the review by Fischbach et al. [2005] identified only 2 cases of agenesis of the corpus callosum, it was detected in approximately 31% of patients with WAGR syndrome analyzed by Han et al. [2013].

A case of monozygotic twins with WAGR syndrome who presented agenesis of the corpus callosum was described by Brémond-Gignac et al. [2005]. The authors suggested that agenesis of the corpus callosum is associated with haploinsufficiency of PAX6. Abouzeid et al. [2009] identified a PAX6 variant that is possibly associated with optic chiasm atrophy and complete agenesis of the corpus callosum.

Microcephaly was reported in a patient with WAGR who had a 7-Mb deletion in 11p [Lennon et al., 2006]. Peng et al. [2004] transiently exposed Xenopus embryos to alcohol and observed that this exposure led to microcephaly and growth retardation, similar to what occurs in alcohol-fetal syndrome. They also found that there was a reduction in the expression of several key genes for neuronal development, such as pax6, otx2, sox3, sox2, and ncam, with the pax6 gene proving the most vulnerable to the action of alcohol. By stimulating Pax6 expression, it was possible to reverse microcephaly and restore the expression of the other affected genes.

Periventricular leukomalacia was also observed in patient P4, who was born after only 30 weeks of gestation. Prematurity is a risk factor for the development of pathologies of the central nervous system [Licht et al., 2015]. This may partly explain the changes in the central nervous system found in P4 [Kinney, 2006]. However, as demonstrated, several genes that have important functions in the development of the central nervous system are located in the region deleted in the patient. Therefore, we suggest that the changes observed are due to both prematurity and the 4.9-Mb deletion in 11p.

Accessory Spleen

The presence of an accessory spleen was reported in patients P3 and P16. The accessory spleen is characterized by ectopic splenic tissue, which arises from a failure in the splenic masses to fuse during embryonic development [Mohammadi et al., 2016].

One of the several deleted genes in P3 and P16 that can explain this congenital malformation is the WT1 gene, which is expressed in the spleen and has an indispensable function for its formation [Mebius and Kraal, 2005]. Schnerwitzki et al. [2014] analyzed wt1 expression in an animal model, including the alternative isoform, and identified that it is highly expressed in the spleen.

Another gene also expressed in the spleen and deleted in P3 is MUC15 [Pallesen et al., 2002], belonging to a family of glycoproteins that make up the main elements of mucus [Oh et al., 2015]. Therefore, the deletion of MUC15 and mainly WT1 may be the cause of the accessory spleen found in P3 and P16.

The deletion breakpoints are not recurrent; the most extensive deletions may extend to the telomere or to the locus of Potocki-Shaffer syndrome [Lacombe et al., 2013]. In general, it is observed that deletions of up to 3 Mb generate mild clinical manifestations, and as the size of the deletions increases, patients have more severe clinical manifestations. However, the severity of the phenotype depends not only on the size of the deletion, but also on the genes mapped to the deleted segments.

Conclusion

In summary, in the present study literature data of selected cases diagnosed with 11p13 deletion through CMA were analyzed. This vast literature analysis allowed us to suggest genes that may be associated with certain characteristics expressed in the evaluated patients. Nevertheless, the disagreement between the genotype and the phenotype in a patient can be explained by the incomplete penetrance observed, for example, in the development of Wilms' tumor, which normally has sporadic origin, but can be inherited in an autosomal dominant manner. Variable expressivity, a concept that has an intimate relationship with incomplete penetrance, also plays an important role, being characterized by the different phenotypic manifestations of the same genotype.

We emphasize the necessity for further investigations to confirm the role of the proposed genes. Finally, this study identified the first patient with a PAX6 partial deletion who does not present any eye anomaly conflicting with our knowledge of PAX6 function and opening a new set of questions about the functional activity of the deleted and remaining PAX6 region observed in this patient.

Statement of Ethics

The study was approved by the University of Brasília Ethics Committee (n. 60137716.0.0000.5558), which conforms to the Declaration of Helsinki standards. The parents gave written informed consent regarding the molecular study and image use.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was partially supported by CAPES, CNPq, and FAPDF.

Author Contributions

Conceptualization: J.F.M., S.F.O., A.P.-T. Formal analysis: V.S.S., G.C.R.C., C.P.O., P.N.M. Funding acquisition: J.F.M., S.F.O., A.P.-T. Clinical evaluation: B.R.V., M.T.A.S.R. Writing, review, and editing: all authors.

Data Availability Statement

All data analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.