Abstract
Background
Epiphyseal, Vertebral, Ear, and Nose (EVEN)‐PLUS syndrome is a rare condition characterized by the involvement of the Epiphyses, Vertebrae, Ears, and Nose, plus other associated findings, due to pathogenic variants in the HSPA9 gene. Due to the sparse number of patients, the clinical phenotypic spectrum is not clear.
Methods
We report two patients with pathogenic HSPA9 variants from a Chinese family. Besides the core clinical features of EVEN‐PLUS syndrome, the two cases had seizures, developmental delay, and basal ganglia lesions in cerebral MRI. We also reviewed the previously published reports of patients with biallelic pathogenic HSPA9 variants.
Results
Together with the presented cases, 12 cases (9 females) were identified from 6 relevant research items for analysis. All patients had synophrys or arched eyebrows, hypoplastic or dysplastic ears, hypoplastic nasal bone, and dysplastic femoral head. Other specific craniofacial features (such as triangular nares), abnormal skeletal presentations (such as bifid femur, dysplastic epiphyses at the knee, dysplastic acetabula, delayed ossification, short stature, vertebral clefting, scoliosis, and dislocated patellae), congenital heart defects, and renal alterations are common clinical features. Two patients had seizures and basal ganglia lesions in cerebral MRI. Infrequent features, such as aplasia cutis, short thorax and sternum, and widely spaced nipples, are also observed in the syndrome. Thirteen variants associated with EVEN‐PLUS syndrome have been reported.
Conclusions
HSPA9 gene mutations should be suspected in all cases with specific craniofacial features, abnormal skeletal presentations, congenital heart defects, and renal alterations. Seizures and basal ganglia lesions are a new phenotype of EVEN‐PLUS syndrome.
Keywords: basal ganglia lesions, epilepsy, EVEN‐PLUS syndrome, HSPA9
HSPA9 gene mutations should be suspected in all cases with specific craniofacial features, abnormal skeletal presentations, congenital heart defects, and renal alterations. Seizures and basal ganglia lesions are a new phenotype of Epiphyseal, Vertebral, Ear, and Nose‐PLUS syndrome.
1. BACKGROUND
The Epiphyseal, Vertebral, Ear, and Nose (EVEN)‐PLUS syndrome is an extremely rare autosomal recessive inherited disease, which is characterized by EVEN dysplasia, plus associated findings (Royer‐Bertrand et al., 2015). The syndrome has a prenatal onset due to defects in the HSPA9 (MIM: #616854) gene, which encodes heat shock protein family A member 9. The protein is a mitochondrial chaperone protein, and it has been linked to many different developmental processes and phenotypic presentations, such as oncogenesis, neurodegeneration, protection from oxidative stress, hematopoiesis, and viral replication (Dores‐Silva et al., 2015).
In 1999, two siblings were reported with an unusual combination of malformations including epiphyseal dysplasia, vertebral dysplasia, and ear anomalies, and the name “EVE (Epiphyseal, Vertebral, and Ear) dysplasia” was proposed (Amiel et al., 1999). Subsequently, EVEN‐PLUS syndrome was first described in 2015 when three patients with the condition were described (Royer‐Bertrand et al., 2015). The syndrome is characterized by its involvement of the EVEN, PLUS other associated findings.
So far, 10 individuals have been reported in the scientific literature with EVEN‐PLUS syndrome and biallelic pathogenic variants in HSPA9 (Amiel et al., 1999; Li et al., 2022; Nagrani et al., 2018; Pacio‐Miguez et al., 2022; Royer‐Bertrand et al., 2015; Younger et al., 2020). The aim of the present work is to report two additional sibs in a Chinese family showing the distinctive neurological phenotype of EVEN‐PLUS syndrome, and it helps to expand and further delineate the features associated with this disorder. In addition, we review the clinical features of these cases and those reported in the literature to refine the phenotype associated with this rare disorder.
2. CASE PRESENTATION
2.1. Ethical compliance
This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Baoding and Beijing Children's Hospital. Written informed consent was obtained from the parents of the patients.
2.2. Case I
The proband was a 14‐year‐old girl, born after an uneventful pregnancy at term in 2006 (Figure 1). Parents are nonconsanguineous and healthy. Her birth weight was 3250 g, without a history of hypoxia‐ischemia and bilirubin encephalopathy.
FIGURE 1.
Pedigrees of affected family (a) and posture and facial photographs of patients 1 (b–f) and 2 (g–j) at the age of 14 years and 5 years and 10 months, respectively. Abnormal standing posture (b, c). Flat facies, microtia, synophrys, and hypoplastic nose.
She was admitted to the Neurology Clinic of Baoding Children's Hospital due to “developmental delay since infancy, abnormal posture and gait for 12 years and 6 months, and intermittent seizures for more than 5 years.” Infant development milestones were delayed. In the motor area, she acquired head control at 3 months, turned over at 5 months, sat at 8 months, and walked alone at 1 year and 6 months. The patient always had an awkward posture and gait since walking alone. The left lower extremity was spasticity and abducted, when walking or running, but both upper limbs could exercise normally. The abnormal posture did not progressively worsen. In terms of intelligence, she could point out simple objects and pronounce “baba, mama” sound, although she could not understand and execute simple instructions at 1 year and 6 months. She could only speak some simple sentences at 4 years old. She could not communicate or play with children of the same age.
At 5 years and 4 months, she had worsening of the abnormal walking posture and gait without an apparent trigger. This manifested as need for assistance with walking and left lower limb dragging, but both upper limbs could move normally. It was not accompanied by infectious symptoms such as fever, cough, vomiting, etc., and no other symptoms such as convulsions, impaired consciousness, and visual impairment. Symptoms peaked at 10 days, gradually recovered, and restored to baseline without special treatment in a month. Urinary organic acid metabolism screening was normal. A lower extremities x‐ray revealed metaphyseal dysplasia. L‐carnitine, fructose sodium diphosphate, and lysine inositol vitamin B12 were used to relieve symptoms; levodopa or diazepam was not used, but her parents stopped the above oral medicine after 2 months. The abnormal walking posture and gait still fluctuated intermittently, worsened, and resolved spontaneously after 3 days to 1 month. At the age of 6 years and 2 months, she was admitted to Beijing Children's Hospital. Brain magnetic resonance imaging (MRI) showed asymmetric high signals on T2‐weighted imaging (T2WI) and T2 Flair in bilateral caudate nucleus, lentiform nucleus, and high signals on diffusion‐weighted imaging (DWI) in the lentiform nucleus (Figure 2). Spine x‐rays showed scoliosis and sacrum lamina fissure. Cerebrospinal fluid (including lactic acid, white blood cell count, level of protein, glucose, and chloride) and other biochemical findings (including plasma electrolytes, calcium, phosphorus, and alkaline phosphatase) were normal. Cardiac and abdominal ultrasound were normal. Plasma ammonia, plasma lactic acid, homocysteine, and re‐analysis of urinary and blood metabolism screening were normal. Electroencephalogram (EEG) showed slow waves in bilateral leads during waking and sleeping phases, and epileptiform discharges in frontal and left posterior area. An electromyography study was performed and excluded any lower motor neuron or muscle involvement. A somatosensory evoked potential test and brainstem auditory‐evoked potential (BSAEP) revealed no abnormalities. The Gesell developmental assessment was performed and indicated severe developmental delay: adaptive ability development quotient (DQ) 25 scores, gross motor DQ 33, fine motor DQ 21, verbal ability DQ 32, and social ability DQ 35. To further clarify the diagnosis, mitochondrial DNA sequencing analysis was conducted and no mutation sites were found with high pathogenicity in mitochondrial DNA. Diagnosed as mitochondrial disease, scoliosis, developmental delay, and epiphyseal dysplasia, physiotherapy was recommended, but her rehabilitation training was not carried out regularly.
FIGURE 2.
Cerebral MRI of patient 1, at the age of 5 years (a), at the age of 6 years (b), and at the age of 14 years (c). Abnormal signals in bilateral caudate nucleus and lentiform nucleus, and series MRI showed caudate and putamen atrophy.
Her first seizure happened at the age of 9 years. Seizures manifested as generalized tonic–clonic seizures, lasting about 1 min before remission, and most occurred during sleep. The frequency of seizures was rare, occurring once in 1 month to several months. Epilepsy was diagnosed, but her parents refused to use any anti‐seizure medications. From the age of 13, seizures were more frequent, 1–2 times a day. With the addition of levetiracetam (30 mg/kg per day) antiepileptic therapy, the frequency of seizures was reduced, to about once in 1 month.
At the age of 14 years, her height was 139 cm (<3th centile), weight 38.5 kg (<3th centile), and head circumference 51.5 cm. Significant midface hypoplasia (Figure 1) included flat facies, microtia, high arched palate, short neck, synophrys, hypoplastic nose, and lateral hair whorls. Abnormal standing and walking posture showed tight hamstring, leg length discrepancy, kyphosis, and scoliosis (Video S1). The neurological physical examination revealed both lower extremities hypertonia, and normal physiological reflexes on both upper and lower extremities. Cranial nerve examination, other muscle strength and tone, sensation, and sphincter function were normal. There were no pyramidal and cerebellar signs.
At the age of 14, cerebral MRI was reexamined and series MRI showed caudate and putamen atrophy (Figure 2).
2.3. Case II
He is the younger brother of the proband. He was born at term (39 weeks) following an uneventful pregnancy and had no history of asphyxia or hypoxia at birth. His birth weight was 3900 g. Since infantile, his motor development milestones were normal but his language and intellectual development had lagged behind that of normal children of the same age. At the age of 3 years, he could only pronounce some onomatopoeic words, such as “baba, mama,” comprehend and follow simple instructions. He received regular rehabilitation therapies with little success. At the age of 4 years, he had a convulsive seizure, which was characterized by loss of consciousness, eyes gazing, clenching of the teeth, and stiffness limbs with shaking, without incontinence, lasting for a few seconds and relieving. A total of 5 intermittent convulsive episodes occurred. Epilepsy and developmental delay were diagnosed at a local hospital. Cranial MRI showed abnormal signals in both basal ganglia (Figure 3). Video EEG showed spike waves and spike‐slow waves emanating from bilateral occipital areas during sleep. His parents rejected to add antiepileptic drugs, and at the last, follow‐up seizure‐free was for 1 year and 2 months.
FIGURE 3.
Cerebral MRI of patient 2, at the age of 8 months (a), and at the age of 4 years (b). Normal MRI (a) and abnormal signals in both basal ganglia (b).
At the age of 5 years and 10 months, his height was 103 cm (<3 th centile), weight 17 kg (3th centile), and head circumference 47 cm. Midface hypoplasia with flat facies, a flat nasal root and poorly developed nasal bridge, bilateral dysplastic ears, high arched palate, short neck and lateral hair whorls (Figure 1). His limbs were normal. Neurological physical examination was normal (Video S2).
His hearing was normal, and BSAEP was normal. Urinary and blood metabolism screening were normal. Cardiac ultrasound, abdominal ultrasound, and renal ultrasound were normal.
2.4. Genetic testing
Whole‐exome sequencing was initially done in case 1 at the age of 14. First DNA was extracted from blood and then targeted gene capture was performed using a custom capture kit. The sequencing was performed to mean >100× Coverage on the Illumina sequencing platform. The obtained sequences were aligned to the human reference genome (GRCh37/hg19) and variant analysis was performed using a set of Bioinformatics Pipeline. Clinically relevant mutations were marked using various published variants in literatures and a set of disease databases “OMIM, ClinVar, HGMD, GWAS, and SwissVar.” Previously not‐reported novel variants found were also analyzed for pathogenicity in vitro. Common variants were filtered based on allele frequency in the 1000 Genome Phase 3, ExAC, EVS, dbSNP147, and 1000 Chinese Genome, etc. Nonsynonymous variants effect was calculated using multiple algorithms such as PolyPhen‐2, SIFT, and MutationTaster2. Only non‐synonymous and splice site variants found in the whole exome panel consisting of a specific set of genes were used for clinical interpretation. Silent variations that do not conduce any change in amino acid in the coding region were not reported.
Compound heterozygous variants in HSPA9 in the proband (NM_004134.7) c.716+2T>C (splicing, maternal) and c.613A>G (p.T205A, paternal) were identified. The former is a novel unreported variant, and the latter is previously reported in another patient (Li et al., 2022). According to ACMG guidelines, the c.716+2T>C variant was classified as variant of likely pathogenic (PVS1 + PM2), and the c.613A>G variant was classified as variant of uncertain significance VUS (PM2 + PM3 + PP3 + PP5). Prediction software from PolyPhen‐2, SIFT, and MutationTaster showed that the c.613A>G variant was likely to be deleterious. The variants were subsequently analyzed by Sanger sequencing in her siblings.
2.5. Radiological skeletal survey
A radiological skeletal survey, following the identification of biallelic HSPA9 alterations in both sibs, was requested for each. The proband showed hypoplastic nasal bones; severe kyphosis and no vertebral clefting were identified. Femoral heads were flattened. Distal femoral epiphyses were dysplastic, with hypoplastic lateral condyle and deep intercondylar notches resembling bifid femora (Figure 4). Her brother showed similar but milder findings, such as femoral heads underossified and flat, distal femoral epiphyses moderately dysplastic without bifid femora, and mild kyphosis.
FIGURE 4.
Radiological findings of patient 1 at the age of 14 years (a–c) and patient 2 at the age of 6 years (d–f). Femoral heads were flattened (a). Distal femoral epiphyses were dysplastic (b), with hypoplastic lateral condyle and deep intercondylar notches resembling bifid femora (c). Femoral heads underossified and flat (d), distal femoral epiphyses moderately dysplastic without bifid femora (e, f).
3. METHODS
3.1. Selection of studies and data extraction
A systematic English and Chinese language literature review was performed to identify previously reported cases with HSPA9 pathogenic variants in the databases of Wanfang, China National Knowledge Infrastructure, PubMed, Web of Sciences, Embase, and Google Scholar using keywords for “HSPA9”, “EVE”, and “EVEN‐PLUS syndrome”, combined with study filters for original research, case reports and case series. We reviewed the reference list of published articles to increase the sensitivity and to select more studies, which we could not retrieve from databases. One of the authors assessed the search randomly, who had not participated in the search previously, to ensure that no reported case has been missed. Full texts of all papers, reports, and documents were extracted. All duplicate items were removed, after screening the titles of documents, year of publication, and names of the authors. Authors independently studied the articles carefully to select relevant articles and exclude non‐relevant ones. Review articles and nonhuman studies were also excluded. Clinical and genetic details of each case, along with family history, were noted on a Microsoft Excel spreadsheet.
3.2. Statistical analyses
Continuous variables were summarized with standard descriptive statistics including means, standard deviation, median, and interquartile ranges while the categorical variables were summarized using frequencies and percentages. Differences among comparison groups in continuous variables were estimated using Student's t‐test for normal distribution variables and using the Wilcoxon rank‐sum test for variables that did not follow the normal distribution. Differences in categorical variables were assessed using the chi‐square test or the Fisher's exact test. A p‐value of <0.05 was considered statistically significant. All statistical analyses were carried out using Statistical Package for the Social Sciences software version 20.0.
4. RESULTS
The systematic search yielded 11 articles. After excluding irrelevant articles, duplicate searches, and including the clinical details of the three cases described above, a total of 10 cases with EVEN‐PLUS (EVE) syndrome were identified in literature from 6 articles (Amiel et al., 1999; Li et al., 2022; Nagrani et al., 2018; Pacio‐Miguez et al., 2022; Royer‐Bertrand et al., 2015; Younger et al., 2020). Detail of the clinical phenotype and genotype in these children is described in Table 1.
TABLE 1.
Clinical features of patients with EVEN‐PLUS syndrome in affected subjects with biallelic HSPA9 pathogenic variants.
| Amiel et al. (1999) | Royer‐Bertrand et al. (2015) | Nagrani et al. (2018) | Younger et al. (2020) | Pacio‐Miguez et al. (2022) | Li et al. (2022) | This study | Total (positive ratio) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient 1 | Patient 2 | Patient 1 | Patient 2 | Patient 3 | Patient 1 | Patient 1 | Patient 1 | Patient 2 | Patient 1 | Patient 1 | Patient 2 | ||
| Sex | F | F | F | F | F | F | M | F | M | F | F | M | 9F/3M |
| Origin | Algerian | Algerian | Korean | Chilean | Chilean | Indonesian | Cherokee‐Caucasian | Spanish | Spanish | Chinese | Chinese | Chinese | |
| Body system | |||||||||||||
| Features | |||||||||||||
| Head/face | |||||||||||||
| Brachycephaly | Unk | Unk | − | − | + | − | + | − | − | − | − | − | 20% (2/10) |
| Eyes | |||||||||||||
| Synophrys and/or arched eyebrows | Unk | Unk | + | + | + | + | + | + | + | Unk | + | + | 100% (9/9) |
| Ears | |||||||||||||
| Hypoplastic/dysplastic external ears | + | + | + | + | + | + | + | + | + | + | + | + | 100% (12/12) |
| Nose | |||||||||||||
| Nasal bone hypoplasia | + | + | + | + | + | + | + | + | + | + | + | + | 100% (12/12) |
| Anomaly of the nares (triangular) | + | + | + | + | + | + | + | − | − | Unk | + | − | 72.7% (8/11) |
| Mouth | |||||||||||||
| Hypodontia | − | − | − | − | + | − | − | − | − | − | − | − | 8.3% (1/12) |
| Chest | |||||||||||||
| Short thorax and sternum | − | − | − | − | − | − | + | − | − | − | − | − | 8.3% (1/12) |
| Widely spaced nipples | − | − | − | − | − | − | + | − | − | − | − | − | 8.3% (1/12) |
| Congenital heart defect | − | − | + | + | + | − | + | − | − | − | − | − | 33.3% (4/12) |
| Abdomen | |||||||||||||
| Hypoplastic Kidney | − | − | − | − | + | + | − | − | − | − | − | − | 16.7% (2/12) |
| Hydronephrosis/vesicoureteral reflux | − | − | − | − | + | − | + | − | − | − | − | − | 16.7% (2/12) |
| Genitalia/anus | |||||||||||||
| Anorectal anomaly | NA | NA | + | − | + | − | + | + | − | − | − | − | 40% (4/10) |
| Unilateral cryptorchidism | − | − | − | − | − | − | + | − | − | − | − | − | 33.3% (1/3) |
| Extremities | |||||||||||||
| Single palmar crease | + | − | − | − | − | − | + | − | − | − | − | − | 16.7% (2/12) |
| Bilateral clubfoot | − | − | − | − | − | − | + | − | − | − | − | − | 8.3% (1/12) |
| Skin/hair | |||||||||||||
| Aplasia cutis | − | + | − | + | + | − | − | − | − | − | − | − | 25% (3/12) |
| Two or three hair whorls | Unk | Unk | − | + | − | + | − | − | − | − | + | + | 40% (4/10) |
| Sparse hair | Unk | Unk | + | − | − | − | − | − | − | + | − | + | 30% (3/10) |
| Skeletal | |||||||||||||
| Dysplastic femoral head | + | + | + | + | + | + | + | + | + | + | + | + | 100% (12/12) |
| Bifid femur | + | + | + | + | + | + | + | + | + | + | + | − | 91.7% (11/12) |
| Dysplastic epiphyses at the knee | + | + | + | + | + | + | − | + | + | + | + | + | 91.7% (11/12) |
| Dysplastic acetabula | + | + | + | + | + | − | + | − | − | + | + | + | 75% (9/12) |
| Vertebral clefting | + | + | + | − | + | + | + | − | − | NA | − | − | 54.5% (6/11) |
| Delayed ossification | + | + | − | + | + | − | + | + | + | + | − | + | 75% (9/12) |
| Short stature | − | − | + | + | + | − | + | − | − | + | + | + | 58.3% (7/12) |
| Scoliosis and/or Kyphosis | − | − | − | − | − | + | + | − | + | − | + | + | 41.7% (5/12) |
| Hip dislocation | − | − | − | + | + | − | + | − | − | − | − | − | 25% (3/12) |
| Dislocated patellae | − | − | − | + | + | + | − | − | − | − | + | + | 41.7% (5/12) |
| 13 pairs of ribs | − | − | − | − | − | − | + | − | − | − | − | − | 8.3% (1/12) |
| Hemivertebra | − | − | − | − | − | − | + | − | − | − | − | − | 8.3% (1/12) |
| Agenesis of the coccyx | − | − | − | − | + | − | − | − | − | − | − | − | 8.3% (1/12) |
| Abnormal and torn meniscus | − | − | − | − | − | + | − | − | − | − | − | − | 8.3% (1/12) |
| Abnormal gait | − | − | − | − | − | + | − | − | − | − | + | − | 16.7% (2/12) |
| Neurologic | |||||||||||||
| Agenesis of the corpus callosum | NA | NA | − | − | + | − | + | − | − | − | − | − | 20% (2/10) |
| Developmental | Normal | Normal | Borderline‐normal | Normal | Moderate developmental delay | Borderline‐normal | Delay | Normal | Normal | Delay | Delay | Delay | Normal 41.7% (5/12), borderline 16.7%(2/12), delay 41.7% (5/12) |
| Agenesis of the septum pellucidum | NA | NA | − | − | − | − | + | − | − | − | − | − | 10% (1/10) |
| Hypotonia | − | − | − | − | − | − | + | − | − | − | − | − | 8.3% (1/12) |
| Hypertonia | − | − | − | − | − | − | − | − | − | + | + | − | 16.7% (2/12) |
| Seizure | − | − | − | − | − | − | − | − | − | − | + | + | 16.7% (2/12) |
| Abnormal signal of Basal ganglia | NA | NA | − | − | − | − | − | − | − | − | + | + | 20% (2/10) |
| Genetic | |||||||||||||
| Biallelic HSPA9 variants | p.Thr362Ile | p.Thr362Ile | p.Val296Ter | p.Arg126Trp | p.Arg126Trp | p.Lys563Ter | p.Leu273Ter | p.Arg126Trp | p.Arg126Trp | p.T294Tfs*3 | c.716+2T>C (splicing) | c.716+2T>C (splicing) | |
| p.Thr362Ile | p.Thr362Ile | p.Tyr128Cys | p.Arg126Trp | p.Arg126Trp | p.Asn149Ile | p.Leu319Phe | p.Ile124Thr | p.Ile124Thr | p.Thr205Ala | p.Thr205Ala | p.Thr205Ala | ||
Abbreviations: NA, not applicable; Unk, unknown.
4.1. Clinical features
A total of 12 cases (9 females and 3 males) from 7 different countries and regions were described. In specific craniofacial and appearance features, all patients had synophrys or arched eyebrows, hypoplastic or dysplastic ears, and hypoplastic nasal bone. Eight cases (72.7%, 8/11) had triangular nares, brachycephaly in two cases (20%, 2/10), single palmar crease in two cases (16.7%, 2/12), and hypodontia in one case (8.3%, 1/12). Nine of the 12 cases had vertebral dysplasia, including six (54.5%, 6/11) in vertebral clefting, five (41.7%, 5/12) in scoliosis, and one in hemivertebra (8.3%, 1/12). In other abnormal skeletal presentations, all patients had dysplastic femoral head, 11 cases (91.2%, 11/12) had bifid femur and dysplastic epiphyses at the knee, nine cases (75%, 9/12) had dysplastic acetabula and delayed ossification, seven (58.3%, 7/12) had short stature, and dislocated patellae, three (25%, 3/12) had hip dislocation, and two (16.7%, 2/12) had abnormal gait. Totally 13 pairs of ribs, agenesis of the coccyx, and abnormal and torn meniscus were described in one of 12 patient (8.3%). In the development milestone, five cases (41.7%) were normal, two were borderline (16.7%), and the others were delayed. Five children had neurologic abnormalities, including agenesis of the corpus callosum and abnormal signal of basal ganglia in two cases (20%, 2/10), seizure and hypertonia in two cases (16.7%, 2/12), and hypotonia in one case (8.3%, 1/12). Congenital heart defects, such as persistent foramen ovale, atrial and ventricular septal defects, and septal aneurysm, were described in four of the 12 patients (33.3%). Renal alterations were observed in three of the 12 patients (25%) with renal ultrasound. Two cases presented vesicoureteral reflux, and two had hypoplastic kidneys with normal kidney function. Four of the 12 patients (33.3%) had anorectal anomaly. Aplasia cutis was observed in three cases (25%). Infrequent features, such as short thorax and sternum, and widely spaced nipples, were described in the same case (8.3%, 1/12).
4.2. Genotypic features
Thirteen variants were reported in association with EVEN‐PLUS syndrome, and one of the 13 variants was located in a splice site (Table 1). Four patients carried homozygous variants in HSPA9 gene, and others carried compound heterozygous variants.
5. DISCUSSION
5.1. HSPA9 gene
HSPA9 gene, located at 5q31.2, encodes a protein with 679 amino acids, which was a highly conserved member of the HSP70 family of proteins, mortalin (Flachbartova & Kovacech, 2013). Mortalin, as a chaperone in the mitochondria, cytoplasm, and centrosome, regulates homeostasis through a wide range of functions, such as vesicular trafficking, iron–sulfur cluster biogenesis, protein folding, and protein translocation (Rosenzweig et al., 2019).
HSPA9 gene has been associated with sideroblastic anemia 4 (autosomal dominant inheritance) and EVEN‐PLUS syndrome (autosomal recessive inheritance). In 2015, in a 4th generation Dutch kindred with mild congenital sideroblastic anemia mapping to chromosome 5q, Schmitz‐Abe et al. (2015) analyzed the candidate gene HSPA9 and identified a heterozygous 2‐bp deletion that segregated with the disease. In the same year, Royer‐Bertrand et al. (2015) reported the EVEN‐PLUS syndrome in 2 Chilean sisters and a Korean girl. Considering its different functions, mortalin might underlie the pathologies of neurodegenerative diseases in Parkinson's disease, but no mortalin mutation significantly associated with Parkinson's disease was uncovered (Szelechowski et al., 2023).
5.2. Clinical phenotype and genotype
The clinical features reviewed in 12 subjects with EVEN‐PLUS syndrome give evidence for a variable clinical spectrum. Twelve cases all had synophrys or arched eyebrows, hypoplastic or dysplastic ears, hypoplastic nasal bone, and dysplastic femoral head, which were not consistent with the core phenotypes of EVEN, because only nine cases had vertebral dysplasia, including vertebral clefting (54.5%, 6/11), scoliosis (41.7%, 5/12) and hemivertebra (8.3%, 1/12). In the special craniofacial features, EVEN‐PLUS syndrome showed a very distinct phenotype with an underdeveloped nose and triangular nares, which were described in 8 cases (72.7%, 8/11). The two siblings reported by Pacio‐Miguez et al. (2022) and case II of our research presented with milder facial features without triangular nares. Abnormal skeletal findings were characteristic and consistent in all subjects and consist mainly of dysplastic femoral heads (100%, 12/12), bifid distal femurs (91.2%, 11/12), dysplastic epiphyses at the knee (91.2%, 11/12), dysplastic acetabula (75%, 9/12), delayed ossification (75%, 9/12), and short stature (58.3%, 7/12). Multiple systems and organs involved were characteristic of EVEN‐PLUS syndrome, such as congenital heart defects (33%, 4/12), gastrointestinal defects (33%, 4/12), genitourinary abnormalities (25%, 3/12), and aplasia cutis (25%, 3/12). Those anomalies were infrequent features but were also part of the phenotypic spectrum associated with HSPA9. Specific examinations, such as cardiovascular ultrasound, urogenital ultrasound, gastrointestinal ultrasound, and radiological skeletal surveys, should be performed in patients with pathogenic biallelic variants in HSPA9, which help us to refine the phenotypic spectrum associated with EVEN‐PLUS syndrome.
In addition to the previously reported features, here we reported two cases with seizures and basal ganglia lesions in cerebral MRI for the first time. Cranial imaging findings of the two siblings were consistent with metabolic or mitochondrial disease, but the specific mechanism was unknown. For the previous study (Moseng et al., 2019), the resulting variants p.R126W and p.Y128C were located on the surface of the mortalin NBD near the binding interface with IDL. These variants disrupted ATP hydrolysis, interdomain linker binding, and thermostability and increased propensity for aggregation, which contributed to the partial understanding of the molecular mechanism for how HSPA9 mutations affect mitochondria functionality and the pathogenesis of EVEN‐PLUS syndrome. The previously reported two sisters carried homozygous variants of p.R126W and presented significantly different central nervous system manifestations, with only one case with developmental delay and corpus callosum dysplasia, and her sister was normal (Royer‐Bertrand et al., 2015). This indicates clinical phenotypic heterogeneity in EVEN‐PLUS syndrome. The abnormal gait of the proband in our study was similar to the case reported by Nagrani et al. (2018). The femur dysplasia and the patellar displacement were the main factors to the abnormal gait. The abnormal gait fluctuated, worsened, and resolved was due to neurologic abnormalities, which could be certified by dynamic changes in a range of cranial MRIs.
We also reported a novel variant in the HSPA9 gene. The patients reported here were a compound heterozygote with the maternally inherited c.716+2T>C variant and the paternally inherited c.613A>G variant. The c.716+2T>C variant was a novel and likely pathogenic variant. Although the reported c.613A>G variant was initially classified as having uncertain significance by the genetic testing laboratory, the clinical picture, segregation, and location of the variants, in silico data suggested that the variant was likely pathogenic.
6. CONCLUSION
This study first reported two cases of EVEN‐PLUS syndrome with seizures and basal ganglia lesions, which expands the spectrum of the clinical phenotype. Core clinical features plus neurological abnormalities should also be considered the disease during diagnosis. In addition, the identification of the new mutation c.716+2T>C expands the spectrum of pathogenic variants in the HSPA9 gene.
AUTHOR CONTRIBUTIONS
Ming Liu and Huanhuan Li made the clinical follow‐up of the family in the clinic, neurological evaluations, and phenotypic descriptions of the siblings. Changhong Ding and Shuhong Ren contributed to the writing of the manuscript and made important comments for its improvement. Ming Liu worked on the manuscript. All authors discussed the results and commented on the manuscript.
FUNDING INFORMATION
This work was supported by the National Natural Science Foundation of China (82001791). This funding had an important role in the collection, analysis, and interpretation of genetic data, but it did not have any role in the design of the study and writing the article.
CONFLICT OF INTEREST STATEMENT
The authors declare that there are no conflicts of interest.
ETHICS STATEMENT
This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Baoding and Beijing Children's Hospital. Written informed consent was obtained from the parents of the patients.
CONTENT FOR PUBLICATION
Written informed consent was obtained from the patient's parents for publication of this Case report and any accompanying images.
Supporting information
Video S1.
Video S2.
ACKNOWLEDGMENTS
We thank the individual included in this study and his parents.
Liu, M. , Li, H. , Ren, S. , & Ding, C. (2024). A new phenotype of EVEN‐PLUS syndrome in a Chinese family and literature review. Molecular Genetics & Genomic Medicine, 00, e2335. 10.1002/mgg3.2335
Ming Liu and Huanhuan Li contributed equally to this study.
Contributor Information
Shuhong Ren, Email: 2579022516@qq.com.
Changhong Ding, Email: dingchanghong@bch.com.cn.
DATA AVAILABILITY STATEMENT
The raw datasets used and analyzed during this study are not deposited in publicly available repositories because of considerations about the privacy or security of human. However, the datasets can be available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Video S1.
Video S2.
Data Availability Statement
The raw datasets used and analyzed during this study are not deposited in publicly available repositories because of considerations about the privacy or security of human. However, the datasets can be available from the corresponding author on reasonable request.