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. Author manuscript; available in PMC: 2025 Nov 24.
Published in final edited form as: Am J Med Genet A. 2025 Oct 11;200(2):521–530. doi: 10.1002/ajmg.a.64280

De Novo Heterozygous ZFX Frameshift Variant in a Female With an X-Linked Neurodevelopmental Disorder

Iftekhar A Showpnil 1, Allison Daley 1, Emily R Sites 2, Shayne M Plourde 1, Jesse M Hunter 1, Dennis W Bartholomew 2,3, April N Lehman 2,3, Daniel C Koboldt 1,3, Rolf W Stottmann 1,3
PMCID: PMC12640707  NIHMSID: NIHMS2124006  PMID: 41074764

Abstract

Germline ZFX variants are associated with an X-linked neurodevelopmental disorder, with 14 males and 16 females reported to date. We describe a 20-year-old female with a heterozygous ZFX frameshift variant, p.(Met666Valfs*2), identified by genome sequencing, previously reported in an affected male. She exhibited motor and speech delays with hypotonia in early childhood, and was later diagnosed with congenital heart defects, autism spectrum disorder, mild intellectual disability, and absence seizures. She further developed sensorineural hearing loss, skin hyperpigmentation, and ophthalmoplegia. Novel phenotypic features included inferior cerebellar vermian hypoplasia, hypoplastic right vertebral artery, aberrant subclavian artery, long palpebral fissures, ophthalmoplegia, skin hyperpigmentation, and a short uvula, expanding the known clinical spectrum. Female carriers of pathogenic ZFX variants demonstrate highly variable expressivity, ranging from apparently unaffected individuals to syndromic presentations. Individuals with heterozygous missense variants often exhibit hyperparathyroidism, suggesting a genotype–phenotype correlation. Reanalysis of published RNA-sequencing data identified 15 ZFX target genes involved in neurodevelopment, suggesting a role for these genes in disease pathogenesis. These findings confirm the pathogenicity of the p.(Met666Valfs*2) variant in the proband and highlight the phenotypic heterogeneity of the disorder in females. Clinical care should include cardiac and endocrine monitoring, with endocrine testing offered to unaffected females carrying missense variants.

Keywords: autism, developmental delay, dysmorphic features, genome sequencing, intellectual disability, MRI findings, rare disease, ZFX

1 |. Introduction

ZFX is an X-linked gene that encodes zinc finger X, a transcription factor with a transactivation domain in the amino terminus and 13 zinc finger domains in the carboxy terminus. Zinc fingers 11–13 are necessary and sufficient for DNA-binding (Ni et al. 2020). ZFX is ubiquitously expressed in all tissue types (Fagerberg et al. 2014) and has been implicated in various cellular pathways. Depletion of ZFX impairs the growth of HEK293T cells (Ni et al. 2020) and has also been linked to inhibition of cell proliferation, cell cycle arrest, and increased apoptosis in a number of cancers (Fang et al. 2012; Jiang et al. 2012; Ma et al. 2015; Song et al. 2018; Wu et al. 2013; Zhu et al. 2013). ZFX is also a known regulator of self-renewal in embryonic and hematopoietic stem cells (Galan-Caridad et al. 2007; Harel et al. 2012). Harel et al. demonstrated that knockdown of ZFX in hESCs results in impaired growth without affecting differentiation potential, while overexpression results in enhanced cell growth and impaired spontaneous differentiation capacity. Mice homozygous for a mutation in Zfx (Zfxnull/null) were smaller, less viable, and had fewer germ cells compared to wild-type mice (Luoh et al. 1997). These findings highlight a role for ZFX in development and growth.

To date, 16 females (aged 8–76 years) from 10 unrelated families with heterozygous ZFX variants have been reported in the literature. These individuals present with a broad spectrum of clinical features, ranging from clinically unaffected to having syndromic neurodevelopmental and dysmorphic features, with a subset of females carrying missense variants presenting with hyperparathyroidism (Guan et al. 2024; Shepherdson et al. 2024). Here, we report novel clinical features in a female harboring a heterozygous ZFX frameshift variant. This variant was previously reported only in an affected male. We expand upon that finding by highlighting potential ZFX target genes that may contribute to disease pathogenesis for loss-of-function alleles. In addition, we perform a comprehensive review of the literature to explore the phenotypic variability of the condition, especially in females, and propose clinical recommendations to guide future practice.

2 |. Case Presentation

The proband is a 20-year-old female of Caucasian ancestry who was first evaluated at 28 months of age in the genetics clinic after a referral for gross motor delay and mild facial dysmorphism (Figure S1). She was born to a 38-year-old gravida 2 para 2 mother via induced delivery at 41 weeks of gestation (weight 3.3 kg, 42nd percentile; length 54.6 cm, 97th percentile), during which she suffered a fractured clavicle. The pregnancy was complicated by gestational diabetes and concerns for Dandy–Walker malformation on prenatal ultrasound and magnetic resonance imaging (MRI). The newborn period was unremarkable except for an inguinal herniorrhaphy at 1 month of age. She exhibited hypotonia and gross motor delay, walking at 26 months. She has a history of stereotypies, such as hand flapping and humming, which decreased as she aged, but tended to occur more frequently when she was tired. She also exhibited episodic heavy breathing along with these behavioral differences. Initial physical exam at 28 months (weight 14.1 kg, 82nd percentile; height 91.5 cm, 73rd percentile; head circumference 51.5 cm, 99th percentile) was remarkable for dysmorphic features. Head and eye examination revealed macrocephaly, long palpebral fissures, epicanthic folds, and mild infraorbital fullness. Her nose was unremarkable. She had a long philtrum, a prominent lower lip, a short uvula, slightly enlarged tonsils, and an asymmetric crying face. Her ears were unremarkable, and her neck was supple. Chest examination revealed a normal anterior–posterior diameter and clear lungs. The heart had a sinus rhythm. The abdomen was soft without masses or organ enlargement. Her extremities were symmetrical with prominent fingertips. Neurologic evaluation at this time did not reveal any localizing signs.

The proband was lost to genetics follow-up for several years but returned at age 12 (weight 34.7 kg, 14th percentile; height 140.1 cm, 5th percentile; head circumference 55.4 cm, 88.8th percentile) with new clinical concerns (Figure 1A). She now had additional diagnoses of autism spectrum disorder, mild intellectual disability (Full-Scale Intelligence Quotient of 61), anxiety, and episodes suggestive of absence seizures. At age 3, speech therapy was initiated by her preschool, focusing on pragmatics and articulation, with a formal evaluation of delayed speech documented at age 5 by a hospital-based speech therapist. Brain MRI at age three had identified an inferior vermian hypoplasia with enlargement of the fourth ventricle in the absence of a posterior fossa cyst. The fourth ventricle did not communicate with any visible cysts. The corpus callosum was well formed, the brain stem appeared grossly normal, the cortical sulcation and gyration pattern appeared normal, and the ventricles were normal in size and configuration. Follow-up MRI at ages 11 and 18 showed these findings to be stable but further revealed a hypoplastic right vertebral artery. She passed her newborn hearing screen on both sides and had a normal audiogram at age 6, but failed a hearing screen in school at age 11. Follow-up testing at a speech and hearing center revealed normal sloping to moderate bilateral sensorineural hearing loss. Following this diagnosis, she used hearing aids for a few years before she stopped wearing them due to social anxiety. Echocardiography identified a bicuspid aortic valve, aortic root dilation (aortic root diameter 2.58 cm, Z-score 2.75), and an aberrant subclavian artery.

FIGURE 1 |.

FIGURE 1 |

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Affected female with a de novo p.Met666Valfs*2 ZFX variant. (A–C) Photographs of the proband depicting the clinical features at ages 12 (A), 18 (B), and 19 (C). Major features included macrocephaly, long palpebral fissures, epicanthic folds, long philtrum, prominent lower lip, asymmetric face, and prominent finger tips. (D) Skin hyperpigmentation on the right distal forearm (left) and mid lower back (right) at age 19. (E) Family pedigree (arrow indicates proband). (F) IGV view of the aligned reads from genome sequencing of the proband, mother, and father depicting the de novo two-base-pair deletion in the proband. (G) Firefly plot of ZFX variants reported in affected individuals in the literature and this study. The recurrent p.Met666Valfs*2 variant identified in the proband is marked with an arrow. (H) Table listing intellectual disability-associated genes that are downregulated after ZFX depletion from HEK293T cells, with sufficient evidence of haploinsufficiency (HI) per ClinGen’s Dosage Sensitivity curation. Dosage sensitivity scores range from 0 to 3 (0—no evidence, 1—little evidence, 2—emerging evidence, 3—sufficient evidence of dosage sensitivity).

At age 18 (Figure 1B; weight 59.8 kg, 64th percentile; height 161.5 cm, 40th percentile; and head circumference 57.5 cm, > 95th percentile), the proband was reevaluated in the genetics clinic after electroencephalogram confirmation of focal absence seizures. Hyperpigmentation of the skin was noted on the right distal forearm, mid lower back (Figure 1D, images acquired at age 19), and posterior left leg (not pictured) on physical examination. The hyperpigmentation was irregular in appearance and seemed consistent with pigmentary mosaicism. Her extraocular movements seemed abnormal, with limited upward and lateral gaze without nystagmus. Follow-up visit at age 19 (Figure 1C; weight 67.1 kg, 80th percentile; height 162.8 cm, 47th percentile; and head circumference 57.2 cm, > 90th percentile) noted ophthalmoplegia with limited upward gaze. Her family history was determined to be noncontributory (Figure 1E).

3 |. Methods

3.1 |. Genome Sequencing and Variant Analysis

Due to suspicion of an underlying genetic disorder, the proband and her parents were enrolled in an Institutional Review Board (IRB)-approved research study for genome sequencing. DNA libraries were prepared using NEBNext Ultra II (New England Biolabs), and paired-end sequencing (2 × 150 bp) was performed using an Illumina NovaSeq 6000 instrument according to manufacturer protocols (Illumina Inc.). Reads were aligned to the GRCh38 reference sequence, and secondary data analysis was performed using Churchill (Kelly et al. 2015). Single-nucleotide variants (SNV) and small insertions and deletions (indels) were called using the Genome Analysis Toolkit (GATK) v.4.0.5.1 (van der Auwera and O’Connor 2020), copy number variants (CNV) were identified using VarScan2 (Koboldt et al. 2013, 2012), and structural variants (SV) were called using Manta v1.6.0 and GRIDDS v2.12.2 (Cameron et al. 2021, 2017; Chen et al. 2016). Common SNVs and indels with minor allele frequency > 0.001 in gnomAD (v4.1.0) were not further considered and followed by the selection of missense variants predicted to be damaging by 75% of 18 in silico pathogenicity prediction tools (including REVEL, MetaRNN, CADD, SIFT, Polyphen2), and all splice site, frameshift, and nonsense variants for further analysis. Resulting variants were assessed for disease etiology, mode of inheritance, phenotypic association, and correlation with the patient’s clinical phenotypes. Rare CNVs and SVs associated with a disease phenotype in the OMIM were also assessed.

3.2 |. RNA-Sequencing

Total RNA was extracted from blood using the Invitrogen MirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Ribosomal RNA depletion and library construction for RNA-sequencing were performed with NEBNext rRNA Depletion Kit (HMR) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA). RNA-sequencing was performed using the NovaSeq6000 instrument (Illumina Inc., San Diego, CA, USA). Reads were mapped to the GRCh38/UCSC hg38 reference sequence using the STAR aligner v2.7 (Dobin et al. 2013).

3.3 |. Phenotypic Correlation Analysis

Phenotypic correlation analysis was performed using clinical data from all individuals with ZFX variants, compiled in Table S1, using the “cor” function in the “stats” R package and visualized using the “ggplot” function in the “ggplot2” R package. Only phenotypes that appear in three or more individuals were considered for this analysis.

4 |. Results

4.1 |. Prior Clinical Testing and Research Sequencing

Following the prenatal suspicion of Dandy–Walker malformation, a karyotype was performed on amniocytes, but did not identify any chromosomal abnormalities (Table S2). At 28 months of age, a PTEN test motivated by the macrocephaly returned negative results. Oligonucleotide microarray at this time also did not identify any chromosomal abnormalities. Following her diagnosis of intellectual disability at age three, fragile X testing was performed and found to be normal. A comprehensive trio exome sequencing test at age 12 and subsequent reanalysis at age 18 did not identify a causal variant in the proband. Given the lack of molecular diagnosis from clinical testing, the proband was referred for genome sequencing under the IRB-approved Congenital Craniofacial Malformations Genetics research study (STUDY00001876). Genome sequencing was performed for the family trio, achieving 54.1×, 42.8×, and 33.6× haploid coverage for the proband, mother, and father, respectively (Table S3). Analysis of SNVs, CNVs, and SVs did not identify any variant of clinical significance. Indel analysis identified a de novo two-base-pair heterozygous deletion in ZFX that results in a frameshift at codon position 666 (Table 1; Figure 1F,G) previously noted in an affected male (Shepherdson et al. 2024). RNA-sequencing of the proband’s peripheral blood revealed expression of both the wild-type and variant alleles at similar variant allele frequencies (Figure S2), demonstrating its escape from random X-inactivation. This variant is absent from large population databases, including gnomAD v4.1.0 (Karczewski et al. 2020), and All of Us (see acknowledgment statement). Loss-of-function variants in ZFX are predicted to be highly intolerant per gnomAD v4.1.0 (pLI = 1.0) with several loss-of-function variants reported upstream of codon position 666 in affected individuals with similar phenotypic features (Figure 1G; Shepherdson et al. 2024). Based on these findings, the ACMG evidence codes PVS1 (null variant in a gene with loss-of-function as a known disease mechanism), PS2 (de novo variant with confirmed parentage and no family history), and PM2 (absent from large population databases) were applied per the standards and guidelines for sequence variant interpretations (Richards et al. 2015), collectively classifying the heterozygous p.(Met666Valfs*2) variant as “Pathogenic.”

TABLE 1 -.

Genomic variant in the proband

Gene Genomic coordinates (GRCh38) HGVS cDNA HGVS Protein Genotype Origin Interpretation
ZFX chrX: 24210954_24210955 NM_003410.4: c.1996_1997del p.(Met666Valfs*2) Heterozygous De novo Pathogenic (PVS1, PS2, PM2)
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4.2 |. Phenotypic Correlation Analysis Demonstrates High Variability in Females

A total of 31 individuals (affected and unaffected) have been reported to carry germline ZFX variants in the medical literature and in this study (Figure 1G; Guan et al. 2024; Shepherdson et al. 2024). A phenotypic correlation with hierarchical clustering of phenotypes (Table S1) separates these individuals into three clusters: syndromic, unaffected, and having hyperparathyroidism, with or without syndromic features (Figure S3). While all known male carriers of hemizygous ZFX variants are affected with previously reported syndromic features, heterozygous female carriers exhibit variable expressivity and incomplete penetrance—6 out of 17 females were unaffected, 4 presented with the characteristic syndromic features, and 7 had hyperparathyroidism with or without syndromic features (Tables 2 and 3). Of the 17 females, 12 harbored a missense variant, and 5 carried a predicted loss-of-function variant (Table 3). Notably, all individuals in both the unaffected and hyperparathyroidism clusters were female, with non-syndromic hyperparathyroidism only appearing in a subset of the females from a single family carrying the heterozygous p.(Y774C) variant (Table 3). The proband clustered with the males, along with three other females carrying either a missense or a loss-of-function variant. Recurring phenotypes in these four females include developmental delays and dysmorphic features that are commonly associated with the core phenotypes of the disorder, with one female in this cluster with a missense variant also exhibiting hyperparathyroidism, hypercalcemia, lethargy, osteoporosis, osteopenia, parathyroid adenoma/hyperplasia, bone pain, and scoliosis. Two females in this cluster also exhibit syndromic features (Tables 2 and S1). Females with loss-of-function alleles, including the proband, either presented with syndromic features without hyperparathyroidism or were clinically unaffected (Table 3). These findings demonstrate a significant clinical variability of observed phenotypes in females harboring a heterozygous ZFX variant, with only missense variants segregating with hyperparathyroidism.

TABLE 2 –

Summary of clinical findings in females with heterozygous ZFX variants. Corresponding frequency of occurrence in males are also noted.

Clinical phenotype Females Frequency in males (14 total)
Frequency (17 total) Variant type
LOF missense
Unaffected 6 (35.3%) 3 3 0 (0%)
Hyperparathyroidism 7 (41.2%) 0 7 1 (7.1%)
Hypercalcemia 4 (23.5%) 0 4 1 (7.1%)
Parathyroid adenoma/ hyperplasia 4 (23.5%) 0 4 0 (0%)
Speech delay 4 (23.5%) 1 3 14 (100%)
Gross motor delay 3 (17.6%) 2 1 14 (100%)
Fine motor delay 2 (11.8%) 1 1 14 (100%)
Mild intellectual / learning disability 2 (11.8%) 1 1 10 (71.4%)
Hypotonia 3 (17.6%) 2 1 11 (78.6%)
Atypical behavior 4 (23.5%) 1 3 7 (50.0%)
Anxiety 3 (17.6%) 1 2 0 (0%)
Attention deficit hyperactivity disorder 3 (17.6%) 1 2 4 (28.6%)
Autism 2 (11.8%) 1 1 5 (35.7%)
Cardiovascular abnormality 3 (17.6%) 2 1 8 (57.1%)
Growth delay 2 (11.8%) 2 0 5 (35.7%)
Macrocephaly 3 (17.6%) 1 2 4 (28.6%)
Facial asymmetry 2 (11.8%) 1 1 1 (7.1%)
Sensorineural hearing loss 2 (11.8%) 1 1 5 (35.7%)
Macrotia 2 (11.8%) 0 2 3 (21.4%)
Recurrent otitis media 2 (11.8%) 0 2 5 (35.7%)
Thick eyebrows/ broad eyebrows/ synophrys 5 (29.4%) 2 3 12 (85.7%)
Epicanthus 3 (17.6%) 2 1 4 (28.6%)
Down slanted palpebral fissures 2 (11.8%) 0 2 6 (42.9%)
Depressed nasal bridge 2 (11.8%) 1 1 3 (21.4%)
Low hanging columella 2 (11.8%) 0 2 3 (21.4%)
Smooth and/or long philtrum 5 (29.4%) 2 3 11 (78.6%)
Palatal abnormality 3 (17.6%) 0 3 6 (42.9%)
Macroglossia 3 (17.6%) 1 2 7 (50.0%)
Enlarged tonsils 2 (11.8%) 1 1 3 (21.4%)
Pointed chin 3 (17.6%) 0 3 6 (42.9%)
Abnormal nail 4 (23.5%) 1 3 10 (71.4%)
Melanocytic nevus 3 (17.6%) 0 3 3 (21.4%)
Macule/ papule 2 (11.8%) 0 2 3 (21.4%)
Umbilical hernia 2 (11.8%) 0 2 6 (42.9%)
Inguinal hernia 2 (11.8%) 1 1 11 (78.6%)
Osteopenia/ Osteoporosis 3 (17.6%) 0 3 1 (7.1%)
Bone pain 4 (23.5%) 0 4 0 (0%)
Scoliosis 3 (17.6%) 0 3 4 (28.6%)
Pectus excavatum 2 (11.8%) 1 1 2 (14.3%)
Sleep abnormality 4 (23.5%) 1 3 5 (35.7%)
Lethargy/ Fatigue 4 (23.5%) 0 4 0 (0%)
Feeding difficulties 2 (11.8%) 1 1 10 (71.4%)
Gastroesophageal reflux 4 (23.5%) 1 3 2 (14.3%)
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TABLE 3.

Summary of heterozygous ZFX variants identified in females.

Proband(s) HGVS Consequence Primary phenotype
This study c.1996_1997del: p.(Met666Valfs*2) LOF Syndromic without HPT
4 (Shepherdson at al.) c.2312C>T: p.(Thr771Met) Missense Syndromic with HPT
Mother and grandmother of 1, (Shepherdson et al.), Guan et al. c.2290C>T: p.(Arg764Trp) Missense Unaffected, unaffected, syndromic with HPT
6I:2, 6II:2, 6II:3, 6II:8, 6III:1, 6III:11 (Shepherdson et al.) c.2321A>G: p.(Tyr774Cys) Missense NSHPT, NSHPT, NSHPT, NSHPT, unaffected, syndromic without HPT
Mother of 8, 9* (Shepherdson et al.) c.2357G>A: p.(Arg786Gln) Missense Syndromic with HPT, syndromic without HPT
Mother of 10 (Shepherdson et al.) c.768dup: p.(Lys257Ter) LOF Unaffected
14 (Shepherdson et al.) c.529dup: p.(Ser177Phefs*12) LOF Syndromic without HPT
Mother of 15 (Shepherdson et al.) c.423_424del: p.(Ser142Ter) LOF Unaffected
Mother of 16 (Shepherdson et al.) c.115_116del: p.(Val39Phefs*14) LOF Unaffected
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*

biological female by birth who is a transgendered male per Shepherdson et al. 2024. Syndromic: syndromic of ZFX-associated neurodevelopmental disorder phenotypes; HPT: hyperparathyroidism; NSHPT: non-syndromic hyperparathyroidism. LOF: loss-of-function.

4.3 |. ZFX Depletion Downregulates Disease-Associated Genes in HEK293T Cells

Deletion of ZFX in HEK293T cells was previously reported to cause differential expression of over 1000 genes (Ni et al. 2020). We therefore sought to identify downstream targets of ZFX that may play a role in neurodevelopment using that differential gene expression dataset. Here, we identified 401 commonly upregulated and 769 commonly downregulated genes between the two clones reported (Figure S4; Tables S4 and S5). Annotation of the commonly dysregulated genes with disease association identified 104 upregulated and 295 downregulated genes associated with a disease in OMIM. Analysis of these genes for dosage sensitivity using ClinGen’s Dosage Sensitivity curations identified only 24 downregulated genes with sufficient evidence of haploinsufficiency as a mechanism of disease (Table S6; Rehm et al. 2015). Of these, 15 are associated with intellectual disability (Figure 1H): OFD1, FGD1, RPS6KA3, FANCB, NHS, CDKL5, PDHA1, SMS, GK, IDS, ALX4, SCN2A, COL3A1, OTX2, and MSL3 (Brown et al. 1994; Fehr et al. 2013; Jones et al. 2016; Kariminejad et al. 2014; McCauley et al. 2011; Riggs et al. 2022; Vandervore et al. 2017; Wolff et al. 2017). Differential regulation of these ZFX target genes may therefore play a part in disease pathogenesis and will require further investigation to establish their role(s) in disease mechanisms.

5 |. Discussion

ZFX is associated with an X-linked neurodevelopmental disorder with core features of developmental delays, intellectual disability, hypotonia, and dysmorphism. Recurrent dysmorphic features identified in our analysis include macrocephaly, thick/broad eyebrows, downslanted palpebral fissures, epicanthus, macrotia, depressed nasal bridge, bulbous nose, low-hanging columella, smooth and/or long philtrum, macroglossia, hallux valgus, and abnormal nail morphology. Affected individuals, however, have been reported to present with variable phenotypes, which we found to be more apparent in females. Of the 17 females reported to carry a heterozygous ZFX variant, including the individual described in this study, 6 females from 5 unrelated families were clinically unaffected, and 4 females from a single family only presented with hyperparathyroidism and were otherwise described to have no developmental or cognitive concerns. The seven remaining females from seven unrelated families presented with syndromic features previously associated with pathogenic ZFX variants, with three of these individuals also presenting with hyperparathyroidism. While all 14 males were reported to have mild to moderate intellectual disability, with 11 of them requiring specialized schooling, only 2 of the affected females displayed mild intellectual disability (this study) or learning disability (Guan et al. 2024), neither requiring special education. In fact, two of the affected females were described as very intelligent by Shepherdson et al., underscoring the phenotypic variability not only between unaffected and affected carriers but also within the small number of affected females. Females carrying loss-of-function variants, including the proband in this study, did not exhibit primary hyperparathyroidism and were either clinically unaffected or presented with syndromic features without hyperparathyroidism. Females carrying missense variants, on the other hand, exhibited significant clinical variability, with individuals reported as clinically unaffected, affected with only hyperparathyroidism, or affected with syndromic neurodevelopmental and dysmorphic features with or without hyperparathyroidism. Females with hyperparathyroidism ranged between 19 and 76 years of age, suggesting no clear connection between age and onset of primary hyperparathyroidism. Given the prevalence of hyperparathyroidism in females in the cohort, parathyroid hormone testing was offered for our proband, which was declined by the family. Since hyperparathyroidism segregated with missense variants, we did not pursue additional endocrine studies in the proband who carries a predicted loss-of-function variant. Nevertheless, endocrine evaluations, including parathyroid hormone testing, should be considered as part of routine evaluation for this disorder, especially for females carrying missense variants in ZFX. Several individuals with hyperparathyroidism were noted to have parathyroid adenoma or parathyroid hyperplasia, conditions known to cause hyperparathyroidism (Bilezikian 2018; Marx 2000). Missense variants in ZFX are therefore likely to play a role in parathyroid development, a mechanism that may not be affected by loss-of-function variants and would require further investigation.

Indel analysis of trio genome sequencing identified a de novo, heterozygous frameshift variant, p.(Met666Valfs*2), in the proband. This variant was not reported in prior clinical exome analysis due to a lack of any gene-disease association at the time. Many of the clinical phenotypes reported in the proband include characteristic neurodevelopmental defects and dysmorphic features noted during early childhood that were consistent with the emerging core phenotypes of ZFX-associated disorder. She was later diagnosed with congenital heart defects and seizures that have been previously reported in a few affected individuals. MRI findings identified inferior cerebellar vermian hypoplasia and hypoplastic right vertebral artery, which are uniquely noted in the proband. Other novel findings in the proband include skin hyperpigmentation, an aberrant subclavian artery, long palpebral fissures, a short uvula, and ophthalmoplegia. Her skin hyperpigmentation appeared irregular and seemed consistent with pigmentary mosaicism. Comprehensive analysis of the genome data did not identify additional variants of interest. These findings not only support the emerging association of ZFX with the X-linked neurodevelopmental disorder but also expand its phenotypic spectrum.

A male patient with a hemizygous ZFX p.(Met666fsVal*2) variant was previously reported in the literature, who was similarly delayed but had presented with additional dysmorphic and cardiac manifestations. The most common cardiac features reported in affected individuals, including the proband in this study, are structural (Table S1), but some affected individuals also display functional anomalies like reduced ventricular ejection fraction. It is therefore recommended that patients establish care from a cardiologist to assess possible cardiac structural defects, with ongoing interval assessment for functional changes.

Consistent with published reports, RNA-sequencing of the proband’s peripheral blood revealed expression of both the wild-type and the variant ZFX allele in the proband, indicating that ZFX escapes random X-inactivation (Schneider-Gadicke et al. 1989; Wainer Katsir and Linial 2019). In addition, X-inactivation studies conducted by Shepherdson et al. on blood samples from affected females in two families demonstrated skewed X-inactivation in both unaffected and affected females, which did not explain the variable clinical presentation among female carriers. Given these findings, X-inactivation studies were not performed on the proband in this study.

Including this study, 31 individuals from 18 unrelated families have been reported to carry a germline ZFX variant in the medical literature—14 males and 17 females. Of these, 20 carry one of four missense variants and 11 carry one of seven loss-of-function variants. The missense variants cluster in zinc finger motifs 12 and 13 of ZFX that are known for their involvement in DNA-binding. Overall, several variants appear in more than one family: R764W (c.2290C>T) in Families 1 and 2; T771M (c.2312C>T) in Families 3–5; Y774C (c.2321A>G) in Families 6 and 7; R786Q (c.2357G>A) in 8 and 9, and M666Vfs*2 in 13 and the proband in this study (Guan et al. 2024; Shepherdson et al. 2024). Interestingly, epigenetic and transcriptomic studies conducted by Shepherdson et al. demonstrated that the missense variants do not have a major effect on the DNA-binding capacity of the protein but instead alter transcriptional activity. The seven reported loss-of-function variants, on the other hand, are predicted to result in the loss of crucial zinc finger motifs involved in DNA-binding, affecting target gene expression.

Deletion of ZFX in HEK293T cells has been shown to disrupt the regulation of numerous target genes (Ni et al. 2020). Reanalysis of this data identified 15 genes (11 on the X chromosome) downstream of ZFX that are implicated in neurodevelopment (Figure 1H). Of note, pathogenic variations in OFD1 are associated with X-linked recessive Simpson–Golabi–Behmel syndrome (MIM#300209), which was considered in the differential diagnoses for several of the affected individuals described in Shepherdson et al., likely due to its high phenotypic similarity to the ZFX-associated intellectual development disorder (Budny et al. 2006; Shepherdson et al. 2024). Interestingly, cerebellar vermian hypoplasia has been observed in X-linked recessive Joubert syndrome patients with OFD1 mutations (Aljeaid et al. 2019; Bisschoff et al. 2013; Coene et al. 2009; Thauvin-Robinet et al. 2013). However, the typical MRI findings of molar tooth sign, thickening of the superior cerebellar peduncles, and enlarged cisterna magna in Joubert syndrome patients are not noted in individuals carrying ZFX variants. Given that hypomorphic OFD1 mutations are known to cause Joubert syndrome and Simpson–Golabi–Behmel syndrome (Budny et al. 2006; Coene et al. 2009), a similar mechanism may be at play where ZFX depletion results in reduced OFD1 expression and subsequent transcriptional activity. Recognizing that the in vitro ZFX knockout studies in HEK293T cells may not be a perfect disease model, these findings propose a hypothesis that mis-regulation of ZFX target genes in individuals carrying a loss-of-function ZFX variant may lead to a disease state. Further investigation is therefore necessary to establish a disease mechanism and identify potential therapeutic targets for this rare genetic disorder.

In summary, males harboring hemizygous ZFX variants present with neurodevelopmental defects and dysmorphic features, whereas females carrying heterozygous ZFX variants exhibit variable expressivity and incomplete penetrance, reflecting X-linked inheritance with sex-specific phenotypic outcomes. Dysregulation of ZFX targets like OFD1 may contribute to disease in loss-of-function cases. Due to the multi-system involvement seen in this disorder, clinical care should include cardiac and endocrine assessments, with parathyroid hormone testing also offered to unaffected females with missense variants. Furthermore, given the potential for phenotypic overlap with other neurodevelopmental disorders, genome sequencing is recommended as a first-tier diagnostic approach to enable accurate detection of ZFX-related disorders.

Supplementary Material

Supplement
Supplemental tables

Additional supporting information can be found online in the Supporting Information section. Table S1: Detailed clinical phenotypes in individuals with ZFX variants reported in the literature and in this study using HPO terms. Table S2: Prior clinical testing. Table S3: QC metrics from genomic sequencing of the family trio. Table S4: Genes commonly upregulated in both clones of ZFX-depleted HEK293T cells in Ni et al. (2020). Table S5: Genes commonly downregulated in both clones of ZFX-depleted HEK293T cells in Ni et al. (2020). Table S6: Genes downregulated by ZFX knockout in HEK293T cells with sufficient evidence of haploinsufficiency (HI) per ClinGen’s Dosage Sensitivity curation. Figure S1: Low resolution polaroid photographs at age 28-months scanned from the proband’s paper chart. Figure S2: RNA-sequencing tracks, generated from proband’s blood sample, displaying the 2 bp heterozygous deletion in the expressed transcripts (supported by 25/55 reads). Figure S3: Correlation heatmap showing Pearson correlation of all phenotypes noted in ZFX-affected individuals in the literature and this study. “1” through “16” refers to the affected individuals reported in Shepherdson et al. (2024). “Proband” refers to the proband reported in this study. Individual 9 was noted to be a biological female who is now a transgendered male (Shepherdson et al.). Guan et al. refers to the single female patient presented in the report by Guan et al. (2024). MGM: maternal grandmother, Mo: mother, m: male, f: female, ms: missense, lof: loss-of-function. Figure S4: Number of genes dysregulated by ZFX knockout in HEK293T cells in Ni et al. (2020). (A) Genes upregulated by ZFX knockout in two clones. The 401 commonly upregulated genes are listed in Table S3. (B) Genes downregulated by ZFX knockout in two clones. The 769 commonly downregulated genes are listed in Table S4.

Acknowledgments

We sincerely thank the patient’s family for their participation in this study. We gratefully acknowledge All of Us participants for their contributions, without whom this research would not have been possible. We also thank the National Institutes of Health’s All of Us Research Program for making available the cohort data examined in this study. This work was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award number R01DE027091.

Funding:

This work was supported by the National Institutes of Health (R01DE027091).

Footnotes

Ethics Statement

This study was performed in accordance with the ethical standards of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) within the Steve and Cindy Rasmussen Institute for Genomic Medicine at Nationwide Children’s Hospital, Columbus, Ohio. The family was enrolled in STUDY00001876: Congenital Craniofacial Malformations Genetics. Written informed consent was obtained from all study participants for enrollment into the IRB approved study, which includes consent for publication of results. A written photo consent was obtained to publish photographs highlighting the clinical features described in the proband.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study will be openly available in https://www.ncbi.nlm.nih.gov/gap/ at dbgap.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement
NIHMS2124006-supplement-Supplement.pdf (1.2MB, pdf)
Supplemental tables
NIHMS2124006-supplement-Supplemental_tables.xlsx (81.8KB, xlsx)

Data Availability Statement

The data that support the findings of this study will be openly available in https://www.ncbi.nlm.nih.gov/gap/ at dbgap.

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