Weatherall et al. (2) were the first to discover a link between hemoglobin H (Hb H) disease and intellectual disability. Wilkie et al. (1) established clinical diagnostic criteria for this condition in 1990, which included severe intellectual disability, microcephaly, developmental delay, characteristic craniofacial malformation, and prominent features of hemoglobin (Hb) H inclusion. The X-linked nuclear protein (XNP)/ATRX gene was isolated in 1994 (3). It was reported that ATRX mutated in 13 patients with alpha-thalassemia/intellectual disability syndrome (ATR-X) syndrome by Gibbons et al. (4) in 1995. Studies provided a more complete picture of the clinical phenotypes of this disease, and it was found that several patients with the identical genotypic configuration had a comparable clinical phenotype but did not have alpha-thalassemia (named for MRXHF1). It has become clear that there are few sine qua non for diagnostic features, the diagnosis should be confirmed by the identification of variants in the ATRX gene.

The human ATRX gene is located in chromosome Xq13.1–q21.1. This transcript of ATRX (NM_000489.6) has 35 coding exons, a transcript length of 11,165 bps, and a translation length of 2,492 residues. The transcriptional regulator ATRX protein (UniProtKB—P46100) encoded by ATRX is strongly expressed in the brain, white blood cells, and skeletal muscle (1). The ATRX protein is a member of the SNF2 family of chromatin remodeling factors, which is involved in chromatin remodeling epigenetic regulation of gene transcription (5). In general, ATRX protein is mainly enriched in telomere, subtelomere, and centromeric repetitive sequence and centromeric tandem repeats. The disruption of these activities may lead to developmental abnormalities associated with the disease.

In this study, a novel hemizygous splicing variant of the ATRX gene was identified in a Chinese boy with MRXHF1. We conducted a literature systematic review to summarize previously reported clinical phenotypes and genetic variants of MRXHF1 according to current diagnostic criteria.

A 3-year-old boy presented with developmental delay and feeding difficulties after birth, with no risk factors that occurred in the developing fetal or infant brain. His family history was not notable. His mother's history of pregnancy was normal. Delivery was at 38 weeks gestation. His birth weight was 3.25 kg, height 50 cm. He did not achieve the normal milestones for his age. Until now, his height is 89.7 cm (<3rd percentile), weight 10.5 kg, and head circumference 43.7 cm (<3rd percentile). He could sit and crawl for a while, but could not stand or walk. He had no response to sounds or simple verbal commands and could not even say simple words. The boy presented with developmental delay, small stature, open mouth with drooling, underdevelopment of tooth, hypotonia, paresthesia, and behavioral disorders in the form of hyperactivity, aggression, and mild facial features. There was no anemia, hepatosplenomegaly, and urogenital abnormalities. The development quotient (DQ) was <20 and adaptive behaviors were extremely impaired. The ECG was normal and the MRI of the brain revealed unremarkable. The patient's hearing was normal and the ophthalmological findings showed no abnormalities. Complete blood count (CBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and hemoglobin A1c (HbA1c) were normal. Hemoglobin electrophoresis showed alkali-resistant HB determination 0.4%, HbA2 2.4%, and HbA 97.2%, and Hb-H inclusion bodies were not detected. The metabolic screening by mass tandem spectrometry and gas chromatography was negative. Laboratory tests, including thyroid function tests, toxoplasmosis, rubella, cytomegalovirus, herpes simplex, and HIV (TORCH) screen, blood ammonia, and lactate, all revealed no abnormal results. After 6 months of rehabilitation therapy and physical therapy combined with speech and cognitive training, there was no significant improvement in neurological function. Because of delayed motor skill milestones and severe intellectual disability, genetic screening in the proband's family for inherited diseases was recommended.

The parents and the patient signed informed consent for genetic analysis. Our legal ethics committee approved this genetic study. The DNA was extracted from the peripheral blood of the proband and phenotypically normal parents for whole-genome sequencing (WGS). Sanger sequencing was used for further verification. The total cellular RNA was isolated from the patient and his mother's peripheral blood for RT-PCR. The DNA was extracted from the patient's mother and maternal grandparents for X-chromosome inactivation (XCI) analysis. We finally identified a hemizygous intronic variant (c.5786+4A>G; NM_000489.6) in the ATRX gene, which has not been reported previously and registered in several variants databases including 1,000 Genomes, gnomAD, dbSNP, HGVD, and ClinVar. Cosegregation analysis was performed among family members. The results of the Sanger sequencing indicated that c.5786+4A>G was inherited from the mother and maternal grandmother. According to the in silico analysis of mutational sequences with MaxEntScan, GTAG and dbscSNV3 showed that the splicing site variant c.5786+4A>G was deleterious and affected the donor site of the entire exon 24. The results of RT-PCR revealed that a proportion of the transcripts of ATRX from the patient lost the entire exon 24, and the mother was normal (Figure 1). The XCI study demonstrated that the carrier mother showed extreme skewing in XCI (Figure 2). According to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for the interpretation of sequence variants (6), the variant was likely pathogenic (PS3+PM2+PP3+PP4).

We searched the PubMed database, Human Gene Variant Database (HGMD), and Online Mendelian Inheritance in Man (OMIM) using “MRXHF1 syndrome,” “ATR-X syndrome,” and “ATRX” as keywords. The search time was from the establishment of the database to November 1, 2021. Previous studies with ATRX variants and their clinical characteristics were included in this review. Nineteen documents were retrieved (7–25). A total of 25 MRXHF1 patients without alpha-thalassemia carrying ATRX gene variants were summarized in Table 1. A total of 21 ATR-X patients with alpha-thalassemia are summarized in Table 2. The most common clinical presentations of MRXHF1 were profound intellectual disability (25/25, 100%), characteristic facial features (24/24, 100%), skeletal abnormalities (14/15, 93%), cardiac defects (15/20, 75%), and genital abnormalities (12/18, 67%). The reported variants were listed in Table 3.

The ATRX-related diseases have emerged as a prominent syndrome among the many X-linked intellectual disability syndromes. Alpha-thalassaemia was previously considered as a feature that distinguishes ATR-X syndrome from the allelic disease (26–30). Although alpha-thalassaemia is commonly present, some patients with the ATRX gene variants do not express this symptom, which showed a wide spectrum of other pathological features. Genetic variants of ATRX are associated with a variety of diseases including ATR-X, MRXHF1, and alpha-thalassemia associated with myelodysplastic syndromes (ATMDS) (OMIM#300448). The ATR-X syndrome is an allelic disorder with the addition of alpha-thalassemia and Hb H inclusion bodies. The ATR-X syndrome and MRXHF1 are both X-linked recessive disorders caused by ATRX germline mutations. The ATMDS is in contrast due to ATRX gene somatic mutations in blood cells presenting more severe alpha-thalassemia.

Here, we reported a 3-year-old boy with a c.5786+4 A>G ATRX gene variant that resulted in moderate to severe phenotypic manifestations. The main characteristics were intellectual disability, severe developmental delay, feeding difficulties, behavioral problems, and hypotonia. The mother was a phenotypically normal carrier. The XCI studies showed that the mother had extremely skewed XCI, which indicated preferential expression of the paternal and inactivation of the maternal X chromosome carrying the ATRX variant. The RT-PCR analysis showed that a proportion of the transcripts of ATRX from the patient lost the entire exon 24, and the mother was normal. The exon 24 of ATRX has the residue conservation of the 30 amino acids (from p.1900 to p.1929), which is located at the C-terminal of the ATRX protein. Hence, it is tempting to speculate that the loss of exon 24 led to ATRX protein truncation and corrupted protein function, which may be the pathogenesis of the disease in this family.

Recent studies reported that a large majority of the disease-associated variants were concentrated in the ATRX-dnmt3-dnmt3l (ADD) (50%) and helicase motifs (30%). To date, more than 150 variants have been described worldwide in ATRX. Missense mutations are more common than other types of variants (31).

Gibbons et al. (31) analyzed the genotype-phenotype relationship in ATR-X syndrome from four aspects. Compared with the helicase region, mutations in the ADD domain produced more severe and permanent psychomotor impairment, usually preventing patients from walking and language acquisition; while the C-terminal may play a special role in the genitourinary system (19). The N- and C-terminus mutations of ATRX protein may cause a milder phenotype of alpha-thalassemia. In addition, researchers found the identified defects in the ATRX-null developing brain were intimately linked to microcephaly phenotype in epigenetic etiology studies of ATR-X syndrome (32) and ATRX protein played an important role in learning and memory (33). It might provide an explanation for the extremely severe intellectual disability observed in a subset of ATRX-related disease syndrome.

Recently, somatic mutations in the ATRX gene have been detected in osteosarcoma (34, 35), pancreatic neuroendocrine tumors (PanNets), glioblastoma multiforme, diffuse intrinsic pontine glioma (DIPG), and neuroblastoma (NB). It is worth noting that if all patients were diagnosed with osteosarcoma at a later age, the symptoms and signs were not the same. However, it is unclear whether there is an association between osteosarcoma and germline ATRX mutations. It has been reported that the ATRX gene had a positive effect on transcription as the Ngln4X gene, a known autism-related gene (36, 37). It is inferred that the ATRX-related diseases and ASD may share phenotypic commonality and mechanism, more research is needed to confirm this hypothesis.

Overall, in this case, in addition to the above symptoms, there are obvious feeding difficulties and gastrointestinal symptoms. These symptoms have been reported in other cases (17, 38). Furuta et al. found that gastrointestinal disorders were closely related to intellectual disability, cerebral palsy, epilepsy, and other neurodevelopmental disorders. In other words, neurological/immune disorders may affect the function of multiple organ systems, including the gastrointestinal tract (39). For neurodevelopmental disorders, we should pay attention to the early feeding status, such as persistent feeding difficulties, which may play an important role in the diagnosis and treatment of the disease. After the exclusion of organic diseases of the digestive tract, such as delayed motor/language development indicators, we should go to the neurodevelopment department in time.

The studies involving human participants were reviewed and approved by Medical Ethics Committee of Yiwu Maternity and Children Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

YC wrote the main manuscript text and carried out the molecular genetic experiments. CH and XY prepared the clinical data and imaging data. KW contributed to the checking of the revision, genetic evaluation, and gene databases analysis. JW and HW critically revised the manuscript. All authors reviewed, read, and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.