Deleterious de novo variants of X-linked ZC4H2 in females cause a variable phenotype with neurogenic arthrogryposis multiplex congenita

2.1 Editorial policies and ethical considerations

The study was carried out in accordance with the Declaration of Helsinki and the protocol approved by the local ethical committees for clinical genetic investigations. Written informed consent was obtained for molecular genetic analysis, publication of clinical, radiological data, and photographs from all participants or their legal guardians.

2.2 Genetic studies

DNAs were extracted from peripheral blood, skin fibroblasts, buccal cells, or umbilical cord using standard procedures.

Families 1, 14, 16, and 18 (DECIPHER Patient IDs: 263304, 263305, 260529, 276496, and 296515) were recruited to the Deciphering Developmental Disorders (DDD) study. DNA samples were analyzed by the Wellcome Sanger Institute using array-CGH and whole exome sequencing (WES; Wright et al., 2015). For families 2, 8, 13, 15, 19, and 24 trio WES was performed as described in more detail in Supporting Information Materials and Methods, and for families 3 and 5 essentially as reported previously (Neveling et al., 2013). For families 4 and 6 all ZC4H2 coding exons were amplified by polymerase chain reaction (PCR) and Sanger sequenced with gene-specific primer pairs previously published (Hirata et al., 2013). Also for family 6, all TYR and OCA2 coding exons were amplified by PCR and Sanger sequenced. For family 7, see, Hennekam et al. (1991) and Hirata et al. (2013). For family 9 WES was performed for the index proband (Figure 1, V:1), his unaffected parents (III:1 and III:2) and two of his affected male relatives (III:5 and IV:2). For family 17 WES was performed for the index proband (Figure 1, II:1) in a research context. For families 3 (umbilical DNA of II:3), 10, 11, 12, 20, and 23 array-CGH was carried out, and for family 12 the breakpoints of the chromosomal deletion were fine-mapped by serial PCR and bidirectional Sanger sequencing (for further details see Supporting Information and Figure S1). For family 21, trio whole genome sequencing (WGS) was performed on peripheral blood DNA from the proband and both parents through the 100,000 Genomes Project (The 100,000 Genomes Project Protocol v3, Genomics England. doi:10.6084/m9.figshare.4530893.v3. 2017). For family 22, trio WGS was performed on saliva DNA at Baylor Genome Center as part of the NIH Gabriella Miller Kids First Research Program. For this family, the GeneMatcher and matchbox nodes of the Matchmaker exchange database were used to obtain collaborations using the search term ZC4H2 (Arachchi et al., 2018; Philippakis et al., 2015; Sobreira et al., 2017). For family 24 trio WES was performed at Fulgent Genetics, US.

The ZC4H2 variants identified by WES and WGS were subsequently confirmed in the patients using Sanger sequencing. Segregation analysis of the variants was performed using standard Sanger sequencing with gene-specific primers and for family 9 using restriction digestion of the specific PCR product (see Supporting Information for more details).

The novel ZC4H2 variants identified through this study have been submitted to the LOVD database (https://databases.lovd.nl/shared/genes/ZC4H2).

2.3 X-inactivation studies

For X-inactivation studies, DNA extracted from blood lymphocytes or skin fibroblasts was analyzed for the methylation sensitive site of FMR1 exon 1 (Carrel & Willard, 1996), the CAG-repeat of the Androgen Receptor (AR) gene (Allen, Zoghbi, Moseley, Rosenblatt, & Belmont, 1992), or of ZMYM3.

2.4 In silico analysis

The pathogenicity of missense variants was assessed using in silico tools including Combined Annotation Dependent Depletion (CADD, http://cadd.gs.washington.edu/) score (Kircher et al., 2014) and Provean (http://provean.jcvi.org/index.php; Choi & Chan, 2015).

2.5 Extraction of variants and clinical phenotypes from the literature and the DECIPHER database

We established a list of all published ZC4H2 variants reported in the literature as of January 2019 and three cases from DECIPHER, and included available clinical information in our analysis.

2.6 Knockdown and rescue experiments in zebrafish

Zebrafish were bred and maintained according to approved guidelines prescribed by the Committee on Use and Care of Animals at Aoyama Gakuin University (Japan). Zebrafish zc4h2 (GenBank NM_199642) cloned into pCR4-TOPO vector (Invitrogen) was used for cRNA synthesis as previously described (Hirata et al., 2013). Antisense morpholino oligonucleotides (MO) designed against the exon 2-intron 2 splice donor site (MO2) of zebrafish zc4h2 were used for knocking down zc4h2. Zebrafish embryos were injected with 5 ng of MOs at 1–2 cell stages and studied as published before (Hirata et al., 2013). For rescue experiments, the missense variants were introduced into the wild-type mouse Zc4h2 (RefSeq NM_001003916.2) pCS2+ expression construct by site-directed mutagenesis (primer sequences are provided in Table S1). Capped RNA was synthesized using mMESSAGE mMACHINE SP6 kit (Life Technologies) according to the manufacturer's protocol. Capped RNA (100 pg) was coinjected with MO2 (4 ng) into zebrafish embryos at 1–2 cell stages. At 2 days postfertilization, normal zebrafish embryos swim away rapidly (> 2 cm/s) upon tactile stimulation. The number of embryos that exhibited slow swimming (< 2 cm/s) following touch was counted.

2.7 ZC4H2 reference sequence

All ZC4H2 variants reported are based on GRCh37/hg19 and transcript isoform 1 (RefSeq NM_018684.3) with nucleotide (cDNA) numbering using +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1.

3.1 Clinical features in the new patient cohort and in previously published families

We report on 23 novel families and simplex cases with likely deleterious ZC4H2 variants and on one previously published family, family 7 of this study (Hennekam et al., 1991; Hirata et al., 2013). The pedigrees of the novel families are shown in Figure 1 (Fam1–6 and Fam8–24) and of family 7 in Figure S2. Detailed clinical descriptions are presented in Supporting Information Results. In the clinical evaluation, we also included publicly accessible information of three females reported to DECIPHER and all families and simplex cases published to date (Godfrey et al., 2018; Hirata et al., 2013; Kondo et al., 2018; May et al., 2015; Okubo et al., 2018; Zanzottera et al., 2017). A compilation of the main (≥ 30%) clinical features of affected males and females from these 42 families is given in Table 1 and of the additional less common clinical features (< 30%) in Table S2. Percentages are related to the total number of probands who were clinically evaluated for a given feature (positive informative). In total, informative clinical data were collected for 87 out of 105 (83%) probands, including 44 out of 48 (92%) males and 43 out of 57 (75%) females using the Human Phenotype Ontology (https://hpo.jax.org/app/) scoring list. Their ages ranged from 23 weeks of gestation to > 70 years at the time of clinical genetic investigation. Families were recruited worldwide and were of Caucasian and Asian ethnicity.

Prenatal presentation of five fetuses with ZARD (two males and three females), included club foot/feet, rocker bottom feet, fetal hypo/akinesia (mostly at the end of the second or third trimester of pregnancy, > 18–26 weeks of gestation), contractures, AMC, nuchal and/or frontal head edema and (nearly) normal growth parameters (See Videos S1–3 and Figure S3). One affected male fetus presented with hypogenitalism (cryptorchidism and micropenis).

At birth and neonatal age, the clinical genetic diagnosis in males varied from descriptive clinical features, for example, AMC with or without additional clinical features including: Psychomotor developmental delay, short stature, brain atrophy, microcephaly, tetraplegia, congenital general hypotonia, facial weakness-palsy, muscle weakness, and complex spasticity, (flexion) contractures of large and/or small joints, congenital hip dislocation – subluxation, rocker bottom feet, club feet, camptodactyly, and ptosis, to (un)recognizable syndromes, such as cerebral palsy, MCS, WRWF with or without cleft palate, feeding difficulties, laryngomalacia, hyperinsulinemic hypoglycemia, mixed obstructive, and central sleep apnea.

The clinical genetic features in 33 newly identified and published carrier females (from childhood till late adulthood) with a maternally inherited ZC4H2 variant varied from normal phenotype (n = 13) to mild (mainly hand-finger) flexion contractures, borderline to mild ID (n = 20) with facial dysmorphism such as ptosis, strabismus, and long (flat) philtrum. Also, distal muscle weakness, urine (stress) incontinence, anosmia, and walking difficulties which were slowly progressive in a few (n = 3) females in late adulthood were reported.

In marked contrast, the 25 females with a de novo pathogenic variant (from neonatal age till adulthood) of ZC4H2 including Xq11.2 microdeletions showed high variation in clinical presentation and ranged from mildly to severely affected (Table 1; Table S2).

Overall, common clinical features in child- and adulthood in both males and females (30% or more positive informative in probands) included postnatal growth retardation, generalized hypotonia, motor delay, inability to walk, spasticity, hyperreflexia, urinary incontinence, dysarthria-deficit in expressive language, poor or absent speech, ID, drooling, dysphagia, chewing difficulties including oral motor dysfunction, feeding difficulties, facial weakness-palsy, high forehead, high anterior hairline, ocular motor apraxia, strabismus, anteverted nares, microretrognathia, AMC, limited shoulder movement, elbow, wrist contractures, metacarpophalangeal joint contractures, camptodactyly, radial deviation of any finger, knee flexion contractures, equinovarus deformity-club feet, Achilles tendon contracture, distal limb muscle atrophy, or weakness, and micropenis or cryptorchidism in males (Table 1; Table S2, Figures S4 and S5).

Less commonly reported clinical features, which were present in <30% of the probands included short stature, microcephaly (more prominent in males (16/26) than in females (4/30)), round face, low-set (posteriorly rotated) ears, upslanting palpebral fissures, almond-shaped eyes, ptosis, deeply set eyes, pupillary dysfunction, nystagmus, short nose, short philtrum, broad alveolar ridges, high-arched palate, and (submucous) cleft palate (Figures S3 and S4). Furthermore, U-shaped upper lip vermillion/carpshaped mouth and downturned corners of the mouth were reported. A short neck (with limited rotation) was more frequently reported for females with a de novo variant (12/14) than for affected males with an inherited or de novo variant (12/37; Table 1; Table S2, Figure S5). Of note, neonatal respiratory distress, recurrent aspiration pneumonia, and (obstructive sleep) apnea can be a particular concern with apnea more frequently reported in males (11/16) than in affected females with a de novo variant (3/11). In addition reported clinical features included narrow chest, narrow shoulders-thorax (more frequently reported in females with a de novo variant (11/15) than in affected males (18/36); Figure S5), umbilical hernia (Figure S3A, 6.III:1), cervical and/or thoracic kyphosis, scoliosis, narrow pelvis, congenital hip contracture, hip dislocation/subluxation, short limbs, proximally placed thumb, ulnar deviation of any finger, overlapping – proximally placed toe(s), edema of the dorsum of hands and feet (Figure S5), generalized hypotonia, absent speech, epileptic seizures, abnormal cortical gyration, delayed CNS myelination, global brain atrophy, and ventriculomegaly (Figure S6). So far, impaired smell was only reported in carrier females in late adulthood. Upslanting palpebral fissures (Figure S4), arrhythmia, sick sinus syndrome, hypoglycemia, and aggressive behavior were so far only reported in males (Table 1 and Table S2).

Besides the ZARD typical features such as AMC, hypo-/akinesia, club foot/feet and facial dysmorphy, associated sporadic clinical features which can be a clue to clinical diagnosis in probands with ZARD include pupillary dysfunction, optic disk cupping, short neck with limited rotation with narrow chest – thorax – shoulders with limited shoulder movements, malposition of the stomach, diaphragmatic eventration, focal autonomic seizures, electrical status epilepticus during slow sleep (ESES) seizure pattern on electroencephalogram (EEG), pancreatic hypoplasia (and postprandial hypoglycemia), and congenital achalasia (Table S2).

Central and peripheral neurological findings in affected individuals included: ID, poor or absent speech, dysarthria, deficit in expressive language, dysphagia, drooling, and chewing difficulties including oral motor dysfunction, motor delay, inability to walk, spasticity, hyperreflexia, areflexia, and dystonia. Various types of seizures, generalized hypotonia, impaired smell (3/13, adult carrier females), and urinary (stress) incontinence (16/28) were also mentioned.

MRI brain and spine images showed variable and global brain atrophy (11/31; examples are given in Figure S6), delayed CNS myelination (9/30), abnormality of periventricular white matter (4/27), corpus callosum abnormality (7/26), abnormal cortical gyration (5/24), ventriculomegaly (8/20), polymicrogyria, anterior to posterior gradient (1/8 males), tethered cord (3/19), and hydromyelia (2/10 males). Abnormal peripheral nerve conduction was present in one affected (1/7 males). Behavioral phenotypes included emotional lability (5/14) and aggressive behavior (3/5 males; Table S2).

Cardiovascular associated clinical features, for example, arrhythmia (5/13), bradycardia (5/8), (congenital) sick sinus syndrome (5/13), and right ventricular hypertrophy (4/12) were so far reported in affected males only (Table S2).

3.2 ZC4H2 genetic characteristics in the new cohort and global mutational spectrum

Most of the novel families were investigated by WES, WGS and/or array-CGH. For two affected index males (family 4, II:2 and family 6, III:1) it was assumed that the phenotype could be caused by a pathogenic ZC4H2 variant and therefore all coding exons were screened by Sanger sequencing.

A total of eight males from six families (families 1, 3, 4, 5, 9, and 19) inherited the variant from a healthy carrier mother, while three males from three families (families 5, 6, and 9) inherited the variant from a mildly affected mother (Figure 1). For one affected male (family 9, II:5) the phenotype of his mother is unknown. In the moderately affected male from family 18 (Figure 1, II:1 and Figures S4 and S5) and the severely affected male from family 24 (Figure 1, II:1) the missense variant occurred de novo.

In 15 females the variant occurred de novo (Figure 1), including individual II:1 from family 6 who presented with contractures of her metacarpophalangeal finger joints and is otherwise healthy. By contrast, the female fetus of family 3 (Figure 1, II:3) inherited the pathogenic variant from her healthy mother who is a mosaic and carries the variant in about 10% of her cells (Figure S7) and the two mildly affected females from family 9 (Figure 1, II:3 and II:6) carry a maternally inherited missense variant.

A total of 23 variants were identified (Figure 2a,b,c). Thirteen of the 23 were de novo loss-of-function variants (nonsense, splice-site, frameshift, and CNVs) present in affected females and 10 were missense variants identified in affected males which, except for the two de novo variants in affected males (families 18 and 24), were inherited from their mildly affected or asymptomatic carrier mothers. Twenty of the 23 variants were novel including single nucleotide variants (SNVs) and five de novo microdeletions in females. The microdeletions removed the first exon of ZC4H2 but did not affect any adjacent genes. Two missense variants were defined as recurrent. Families 4 and 24 of this study carry a previously reported p.(Arg198Gln) change in an unrelated family (Hirata et al., 2013). Family 18 of this study has a de novo p.(Arg213Trp) variant previously reported in three unrelated families (Hirata et al., 2013; May et al., 2015). Also, in two families in this study (families 5 and 6) the same amino acid (Ala200) was altered. Finally, the proline residue 201 mutated to serine in a published family (Hirata et al., 2013) was mutated to histidine in family 19 of this study.

None of the variants identified in this study were reported in publicly available population databases, including the 1000 Genomes Project database and Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/). Also, there were no deletions reported in the database of genomic variants (DGV, http://dgv.tcag.ca/dgv/app/home?ref=GRCh37/hg19) overlapping ZC4H2 exons, demonstrating that the variants identified in the patients are likely not common polymorphisms and supporting our suggestion that they are deleterious to protein function. All missense variants altered highly conserved amino acids (fully conserved from human to zebrafish; Figure 2d and Figure S8). Also, in silico predictions using CADD revealed high scores (> 22) and using Provean all variants were predicted as deleterious, except for the p.(Lys217Arg) change, which perfectly cosegregated with the phenotype in a large family (Fam9). At the gene level, ZC4H2 is highly constrained with a pLI score of 0.91 and zero known LOF mutations in gnomAD (Lek et al., 2016).

Combined with our results of this study, there are currently 31 unique ZC4H2 variants known in affected males and females, including one de novo X-inversion interrupting ZC4H2 in a male plus 10 de novo Xq11.2 microdeletions in females (Figure 2a). An overview of all variants is provided in Table S3. Thus far, SNVs leading to missense changes cluster in the last exon of ZC4H2, which encodes the zinc-finger domain and the most C-terminal part of the protein.

The X-inactivation pattern in blood or skin fibroblasts in affected females varied from random to skewed and was not useful to predict the phenotype (Figure 1 and Supporting Information Results), for example, one severely affected female fetus showed an Xi ratio of 61:39 in skin fibroblasts (Figure 1, Fam3, II:3). Other mild to severely affected females showed Xi ratios varying between 100:0 and 80:20 (Figure 1) in blood lymphocytes. Previous asymptomatic carrier females reported in the literature showed also a ratio of > 95:5 (e.g., Hirata et al., 2013).

3.3 Zebrafish studies

We investigated the potential effects of the p.(Ala200Val) and p.(His70Gln) missense variants which we identified early on in zebrafish morphants similar to our previous studies (Hirata et al., 2013). Following knockdown of zc4h2, zebrafish morphants showed impaired swimming capability at 2 days postfertilization (27/34, 79% of morphants) due to compromised swimming contraction. This defect could be rescued with wild-type Zc4h2 (7/29, 24% of morphants), but not with constructs carrying the p.(Ala200Val) (27/35, 77% of morphants) or p.(His70Gln) (29/41, 71% of morphants) variants and thus the results strongly supported pathogenicity of the altered amino acids.