Detection of the Heterozygous Recurrent MAX p.(Arg60Gln) Variant in Two Females Confirms and Expands the Phenotypic Spectrum of Polydactyly–Macrocephaly Syndrome

ABSTRACT

A recurrent de novo germline variant in the MAX gene, p.(Arg60Gln), has recently been associated with polydactyly‐macrocephaly syndrome in six unrelated individuals. Affected individuals presented with progressive macrocephaly, post‐axial polydactyly, developmental delay, autistic features and a series of craniofacial, brain, cardiac, ocular, and renal anomalies. Here, we describe two unrelated female probands with the known recurrent MAX variant, c.179G>A p.(Arg60Gln), who presented with the emerging phenotypes of the MAX‐associated syndrome. We also propose that genitourinary abnormalities, including Mayer–Rokitanski–Kuster‐Hauser syndrome in one individual, may constitute an expansion of the known phenotype. These findings contribute to the current knowledge regarding the phenotypic spectrum of MAX‐associated polydactyly‐macrocephaly syndrome.

We identified a recurrent heterozygous MAX c.179G>A:p.Arg60Gln variant in two unrelated females affected with the emerging phenotypes of MAX‐associated polydactyly‐macrocephaly syndrome. We propose that genitourinary abnormalities, including Mayer–Rokitanski–Kuster–Hauser syndrome in one individual, are an expansion of the known phenotypes associated with this syndrome.

1. Introduction

The MYC‐associated factor X (MAX) is a transcription factor that binds the canonical enhancer‐box (E‐box) DNA sequence (CACGTG) and promotes transcriptional activation as a MYC/MAX heterodimer [1, 2]. E‐box sequences are found in the promoters of many c‐Myc target genes. MAX can also homodimerize to bind the E‐box with similar affinities and specificities as the heterodimer [3]. While the MYC/MAX heterodimer binds these sites to act as a transcriptional activator, the MAX homodimer binds these sequences to inhibit transcription, thus regulating the expression of c‐Myc target genes.

Germline variants in MAX have been linked to pheochromocytoma susceptibility [4], but a recent publication described a heterozygous, recurrent de novo MAX 179G>A, p.(Arg60Gln) germline variant in three unrelated males [3]. These individuals had a syndromic overgrowth disorder characterized by progressive macrocephaly and post‐axial polydactyly. Other common features of this disorder included developmental delays, brain abnormalities, autistic traits, visual memory loss, and poor visual attention. Ocular, cardiac and renal involvement were also noted. Three more patients were later reported to share the same MAX p.(Arg60Gln) variant. These individuals exhibited novel craniofacial and cardiovascular phenotypes in addition to the previously identified core features [5, 6]. We present the occurrence of this unique, recently identified genetic disorder in two females with the heterozygous, recurrent MAX p.(Arg60Gln) variant.

2. Methods

2.1. Family 1

Proband 1, the affected individual in Family 1, was previously described as a carrier for a mosaic pathogenic variant in ARID1A NM_006015.6: c.169G>T: p.(Glu57*) that partially explained her ocular abnormalities [7]. Since the primary clinical features described in the individual remained unexplained, the individual was referred for exome sequencing which was performed for the proband and her mother (duo exome sequencing).

2.2. Family 2

Chromosomal Microarray Analysis was performed for the affected individual in Family 2 (Proband 2), followed by exome sequencing for the proband and parents (trio exome sequencing), and analyzed by the Genomics team at the Sheba Medical Center, as previously described [8].

3. Results

3.1. Clinical Presentations

Proband 1 is a Caucasian female with normal growth parameters at birth (Table 1). Fetal magnetic resonance imaging (MRI) revealed a left ureteropelvic junction obstruction, mild right renal pelviectasis, mild right lateral ventriculomegaly, and bilateral post‐axial polydactyly. A brain MRI performed at 5 months of age demonstrated abnormal sulcation in the occipital and parietal lobes, hypoplasia of the corpus callosum, gray matter heterotopia, and abnormal sulcation near the posterior sylvian fissure with possible closed‐lip schizencephaly on the right. At 16 months of age, her height and weight were within normal ranges, although her head circumference was notably enlarged, which was consistent with macrocephaly.

TABLE 1.

Clinical findings.

	This study		Harris et al. [3]			de Oliveira et al. [5]	Gomes et al. [6]
	Proband 1	Proband 2	P1	P2	P3	P1	P1	P2
Demographic information
Age	11y	15y6m	7y1m	9y9m	Deceased	19y	9m	7y
Sex	Female	Female	Male	Male	Male	Male	Female	Male
Ethnicity	Caucasian	Caucasian	N/R	N/R	N/R	N/R	Hispanic	Caucasian
Prenatal
Gestational period (weeks)	37	39	N/R	N/R	N/R	(Term)	38	39
Delivery type	Spontaneous vaginal delivery	Spontaneous vaginal delivery	N/R	N/R	N/R	N/R	Spontaneous vaginal delivery	Spontaneous vaginal delivery
Prenatal manifestations	Polyhydramnios	N/R	N/R	N/R	N/R	N/R	−	N/R
Growth parameters
OFC (cm)	Birth: 35.5 (z = 0.47); 16m: 47.2 (z = 3.34); 6y 5m: 56.5 (z = 4.0); 7y: 57 (z = 4.4)	15y 6m: (> 3SD)	Birth: +1.94 a 7y 1m: +3.4 a	2y 6m: +3.2 a 9y 9m: +3.02 a	Birth: Normal	Birth: 36 (85th percentile) 1m: 40 (97th percentile) 19y: 61.5 (> 99th percentile)	Birth: 34 (z = 0.10), 8m: 66th percentile (z = 0.42)	Birth: unavailable, 7 weeks: 99th percentile (z = +2.5), 7y: 57 (z = +4.5)
Weight (kg)	Birth: 3.11 (z = −0.59); 16m: 8.60 (z = 1.41); 7y: z = 1.59	Birth: 3.5; 15y 6m: 49 (z = −0.50)	N/R	N/R	N/R	Birth: 3.45 (50–85th percentile) 1m: 4.33 (85–97th percentile) 19y: 65.8 (15‐50th percentile)	Birth: 3.1 (z = −0.29), 8m: 14th percentile (z = −1.10)	Birth: 4.8 (z = +2.63), 7y: 85th percentile (z = +1.07)
Length (cm)	Birth: 47.5 (z = −0.98); 16m: 66.4 (z = 0.26); 7yo: z = 0.81	15y 6m: 168 (z = 0.88)	N/R	N/R	N/R	Birth: 49.5 (50th percentile) 1m: 55.5 (50–85th percentile) 19y: 182 (50–85th percentile)	Birth: 50.5 (z = 0.73), 8m: 37th percentile (z = −0.33)	Birth: unavailable, 7y: 71st percentile (z = +0.56)
Clinical phenotypes
Progressive macrocephaly	+	+	+	+	N/A	+	N/A	+
Post‐axial polydactyly	+	+	+	+	+	+	+	+
Developmental delay	+	+	+	+	N/A	N/A	+	+
Intellectual disability	+				N/A	+	N/A	+
Autism/Autistic traits	+	N/A	+	+	N/A	+	N/A	N/R
Ventriculomegaly	+	+	+	−	−	N/A	−	−
Craniofacial	Supernumerary mandibular central incisor	−	−	−	−	Elongated palpebral fissures, protruding and posteriorly rotated ears	Unilateral cleft lip and alveolus, natal tooth, maxillary hypoplasia, low‐set and posteriorly rotated ears	Ankyloglossia, two natal teeth, periorbital fullness, epicanthic folds, thick eyebrows, mild posteriorly rotated ears, mild long philtrum, widely spaced upper incisors
Ophthalmic abnormalities	+	+	+	+	N/A	−	−	−
Genitourinary abnormalities	Left ureteropelvic junction obstruction; mild right renal pelviectasis	Uterine agenesis; rudimentary vagina; small left kidney with single cyst	−	−	Hypospadias; bilateral renal agenesis	N/A	−	Bilateral hydroceles
Cardiovascular abnormalities	−	−	Persistent patent foramen ovale (now closed)	−	Atrial septum defect; single umbilical artery	−	Coarctation of the aorta, secundum atrial septal defect	N/R
Other brain imaging abnormalities	Abnormal sulcation in the occipital and parietal lobes and the posterior sylvian fissure; gray matter heterotopia; hypogenesis of the corpus callosum; cerebellar dysplasia; cleft in the ventral brainstem; scattered foci of periventricular gliosis with a posterior predominance	Shortened corpus callosum	Prominent perivascular spaces in the basal ganglia/peri‐caudate region	Prominent perivascular spaces in the basal ganglia/peri‐caudate region; prominent perivascular spaces posteriorly adjacent to the trigone of the right lateral ventricle	Swollen brain; decreased gray‐white matter differentiation; decreased demarcation of the cortex	−	Mild asymmetry of the lateral ventricles	−
Additional phenotypes			Gastro‐esophageal reflux; 4 phalanges on left thumb; pectus carinatum	Perianal abscesses	Died 1 h after birth; flattened thoracic vertebrae	Long fingers and toes	Triphalangeal thumbs, sacral dimple, subglottic stenosis, mild macrocytic anemia	Triphalangeal thumbs, feeding difficulties, tracheomalacia, mild diastasis recti, sacral dimple, sacrococcygeal teratoma, filar lipoma

Abbreviations: N/A, not applicable; N/R, not reported.

^a Harris et al. [3] reported the head circumference for individuals P1 and P2 in standard deviations relative to the UK_1990 mean for age and sex.

At 6 years and 5 months, Proband 1 was re‐evaluated in the clinic. Her growth parameters were again within normal limits, except for her persistent macrocephaly A SNP chromosomal microarray was negative. However, an intellectual disability and autism gene panel identified mosaicism for a pathogenic heterozygous ARID1A nonsense variant [NM_006015.6: c.169G>T: p.(Glu57*)]. In the absence of any other causal variants, this ARID1A variant was considered diagnostic for Coffin‐Siris syndrome, which partially explained the proband's features [7].

Proband 2 presented for genetic evaluation at 15.5 years of age due to multiple congenital anomalies and behavioral difficulties. Macrocephaly was noted after birth and a brain MRI demonstrated a shortened corpus callosum with ventriculomegaly. Bilateral hand postaxial polydactyly was observed. Imaging of the reproductive system was indicated by amenorrhea and revealed agenesis of the uterus and a rudimentary vagina without dysgenesis of the ovaries. These latter findings were consistent with a clinical diagnosis of Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome. Renal ultrasound and dynamic kidney scans were performed for evaluation of recurrent urinary tract infections (UTIs) and showed a relatively small left kidney with a single cyst.

3.2. Variant Assessment

Sequence analysis of both pedigrees identified a variant in the MAX gene [(GRCh38) g.65078029C>T: NM_002382.5, c.179G>A: p.(Arg60Gln)] which was not found in any other sequenced family members and was presumed to be de novo (Table 2). The MAX p.(Arg60Gln) variant was previously reported in a total of six unrelated individuals with an emerging polydactyly–macrocephaly syndrome [3, 5, 6]. The variant exists in a well‐established DNA binding domain that has two other pathogenic/likely pathogenic missense variants listed in ClinVar [9] (Figure 1C), is absent from the gnomAD v4.1.0 population database [10] and only one variant allele was noted in the All of Us database [11]. This variant is also reported in ClinVar with conflicting classifications of pathogenicity (one likely pathogenic, two uncertain significance).

TABLE 2.

Genomic finding.

Gene	MAX
HGVS	GRCh38/chr14:NC_000014.9:g.65078029C>T; (NM_002382.5):c.179G>A; (NP_002373.3):p.(Arg60Gln)
dbSNP ID	rs2063106020
ClinVar ID	958 620
Genotype	Heterozygous
OMIM phenotypes	(AD) Polydactyly‐macrocephaly syndrome (MIM#620712); (AD) Pheochromocytoma susceptibility (MIM#171300)
Interpretation	Pathogenic (PS3, PM1, PM2, PP3, PP5)

FIGURE 1.

Recurrent heterozygous MAX p.(Arg60Gln) variant in two affected females. (A) Family pedigrees with the affected probands indicated by the dark circle. Mother and father were unaffected in both families. (B) IGV view of aligned reads for the proband and mother's exomes in Family 1 showing the heterozygous MAX variant [(GRCh38) g.65078029C>T: NM.002382.5: c.179G>A: p.Arg60Gln] in the proband. (C) FireFly plot depicting the recurrent MAX p.(Arg60Gln) variant in all reported individuals affected with the polydactyly–macrocephaly syndrome (including this study). The plot also shows all pathogenic/likely pathogenic variants reported in ClinVar for MAX‐associated pheochromocytoma susceptibility.

Prior in vitro studies using the wild type and the p.(Arg60Gln) variant alleles of MAX demonstrated that the variant reduces the binding affinity of homodimeric MAX to the E‐box DNA sequence and leads to more efficient heterodimerization of MAX with c‐Myc [3]. This then leads to dysregulation of c‐Myc target genes, including upregulation of CCND2. Interestingly, CCND2 gain‐of‐function is associated with megalencephaly‐polymicrogyria‐polydactyly‐hydrocephalus syndrome [12], a syndrome that was initially considered in the differential diagnosis for the proband [7]. MAX is also known to be a tumor suppressor susceptibility gene and germline variants in MAX (including the variant presented here) have been shown to predispose carriers of the variant to neural crest‐derived neoplasms, specifically pheochromocytomas and paragangliomas [13, 14]. Fluorescent polarization assays measuring DNA binding affinity by Wang et al. demonstrated that the p.(Arg60Gln) variant in MAX significantly reduced DNA binding (by ~20 fold). Taken together, these findings strongly support a damaging effect of the MAX p.(Arg60Gln) variant. Based on these findings, ACMG evidence codes PS3 (well‐established functional studies demonstrating a damaging effect of the variant), PM1 (located in a critical functional domain without benign variations), PM2 (rare in population databases), PP3 (multiple computational tools predict a deleterious effect), and PP5 (reputable source reports the variant as pathogenic) were applied, classifying the MAX p.(Arg60Gln) variant as “Pathogenic” (Table 2) [15].

4. Discussion

We present here two affected females with a heterozygous, recurrent MAX p.(Arg60Gln) variant. This variant is associated with polydactyly‐macrocephaly syndrome (MIM#620712) based on the findings from previous studies [3, 16]. The individuals reported here not only fit the characteristic features consistent with the emerging core manifestations of this syndrome—post‐axial polydactyly, progressive macrocephaly, developmental delays and/or autistic traits—but also expand the phenotypic spectrum of this newly recognized condition. Novel clinical findings in this report are the prenatal manifestation of polyhydramnios; genitourinary abnormalities including left ureteropelvic junction obstruction, mild right renal pelviectasis, and MRKH syndrome; brain MRI abnormalities including abnormal sulcation, gray matter heterotopia, corpus callosum involvement, and cerebellar dysplasia. While dental abnormalities were not reported in Harris et al., a recent report highlighted two affected individuals with natal teeth [6]. Our finding of the supernumerary mandibular central incisor in Proband 1, along with the findings in Gomes et al., therefore, suggests a role for the heterozygous MAX p.(Arg60Gln) variant in tooth development. While many features of Proband 1 fit those of the MAX‐associated syndrome, her polydactyly was less severe than what is reported in Harris et al. We also identified brain anomalies that were more prominent in this patient than in affected patients reported previously. It is also likely that the mosaic ARID1A variant in Proband 1 has a role to play in the severity of her brain anomalies and visual impairment compared to those observed in the cohort of MAX‐affected individuals. Proband 1's primary clinical features of progressive macrocephaly, ventriculomegaly, post‐axial polydactyly, and genitourinary anomalies, however, were not associated with the well‐established features of ARID1A‐associated Coffin–Siris syndrome and can be attributed to the heterozygous MAX p.(Arg60Gln) variant. Interestingly, while genitourinary problems are not currently reported in the clinical synopsis for the polydactyly‐macrocephaly syndrome in OMIM, one of the three individuals carrying the heterozygous MAX p.(Arg60Gln) variant in Harris et al. was described to have hypospadias and renal agenesis. Of special note, is the finding of major congenital anomalies of the female reproductive system, consistent with MRKH syndrome, in Proband 2 reported herein. MRKH is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a 46,XX karyotype. While its molecular basis was unknown and elusive for decades, several candidate genes have been proposed to play a role, including HNF1B, LHX1, ZNHIT3 and WNT4 [17]. In this regard, our findings might shed new light on the genetic architecture of MRKH and underscore that variants in MAX should also be considered in relevant clinical circumstances. Given the genitourinary anomalies we observe in the affected females in this study, we propose that these defects are a more common feature of the MAX‐associated syndrome than was previously recognized. In conclusion, our findings demonstrate the full penetrance of the heterozygous MAXp.(Arg60Gln) variant in two unrelated affected females, further confirm its pathogenicity, and expand on the phenotypic manifestations of MAX‐associated polydactyly‐macrocephaly syndrome.

Author Contributions

All authors contributed to data acquisition, scientific discussion, variant interpretation, and manuscript review.

Ethics Statement

Our study included two affected females who were referred by their clinician after presenting a phenotype consistent with a rare genetic disorder. This study was performed in accordance with the ethical standards of the Declaration of Helsinki under a research protocol approved by the respective Institutional Review Boards at Cincinnati Children's Hospital Medical Center (Study ID: Next‐Gen Sequencing for Congenital Craniofacial Malformations 2019‐1049) (Proband 1) and Sheba Medical Center (Study ID: SMC‐8668‐21) (Proband 2).

Consent

Written informed consent was obtained from all study participants for enrollment into the IRB approved studies, which (for Study 2019‐1049) include consent for publication of results.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: cge70101‐sup‐0001‐Supinfo1.docx.

Showpnil I. A., Feinstein‐Goren N., Greenbaum L., et al., “Detection of the Heterozygous Recurrent MAX p.(Arg60Gln) Variant in Two Females Confirms and Expands the Phenotypic Spectrum of Polydactyly–Macrocephaly Syndrome,” Clinical Genetics 109, no. 4 (2026): 788–795, 10.1111/cge.70101.

Data Availability Statement

Raw sequencing data for family 1 are available in dbGAP (NIDCR Syndromic Orofacial Clefting, phs002997.v1.p1 “Genetic Analysis of Syndromic Orofacial Clefting”) as part of the R01 study R01DE027091. The mutation diagram in Figure 1C was generated using FireFly v1.1.5 (https://fireflyplot.org) using information from PFAM [18], and the ClinVar database (last accessed on September 25th, 2024). Access to data regarding family 2 is available upon reasonable request from the corresponding author.