StemCell Therapy

Clinical features of patients

Between 2015 and 2017, a total of 177 patients (81 males; median [range] age, 4 [030] years) from 169 families were referred to the TOKAI-IRUD program. All patients registered in this study were new patients, i.e., those who had not been previously analyzed for comprehensive genomic variants; however, several patients have been included in a few subsequent investigations19,20,21,22.

The TOKAI-IRUD program is open to the possibility of accepting any patient. The clinical symptoms of the applicants were global developmental delay (HP: 0001263; n=95, 54%), seizures (HP: 0001250; n=40, 23%), intellectual disability (HP: 0001249; n=29, 16%), muscular hypotonia (HP: 0001252; n=24, 14%), dysmorphic facial features (HP: 0001999; n=17, 9.6%), short stature (HP: 0004322; n=14, 7.9%), microcephaly (HP: 0000252; n=11, 6.2%), and others (n=38, 21%) (Table 1, Supplementary Table S2, and Supplementary Table S3).

In accordance with ACMG guidelines, pathogenic SNVs were identified in 36 (20%) patients. Furthermore, 30 (17%) patients carried SNVs classified as likely pathogenic based on clinical validity assessment and consistency in clinical information and phenotypes with applicable diseases. Among 66 patients with pathogenic or likely pathogenic SNVs, 47 had autosomal dominant genetic disorders, seven had autosomal recessive genetic disorders, eight had X-linked dominant genetic disorders, and four had X-linked recessive genetic disorders (Fig.1).

Patient characteristics and information on detected variants. Each column indicates one patient. SNV single-nucleotide variant, CNV copy number variant, UPD uniparental disomy, AD autosomal dominant, AR autosomal recessive, XLD X-linked dominant, XLR X-linked recessive.

Copy number analysis identified diagnostic duplication/deletion in 11 (6.2%) patients, and these included a 10q26.3 deletion (TOKAI-IRUD-1135 and TOKAI-IRUD-1273), 22q11.2 duplication (TOKAI-IRUD-1236), 5q14.3 deletion (TOKAI-IRUD-1252), 47,XXY (TOKAI-IRUD-1297), 1p36 deletion (TOKAI-IRUD-1301), 7q11.23 duplication (TOKAI-IRUD-1321), 19p13.13 deletion (TOKAI-IRUD-1335), 16p13.3 duplication (TOKAI-IRUD-1337), 17p11.2 duplication (TOKAI-IRUD-1343), and 4p16.3 deletion (TOKAI-IRUD-1475).

ROH analysis identified homozygous regions larger than 10Mb in 105 cases; this included a diagnostic upd(15)pat in 1 patient (0.6%) who was diagnosed with Angelman syndrome (TOKAI-IRUD-1290, OMIM #105830). Furthermore, UPD of a whole chromosome was identified in 2 (1.1%) patients [upd(2)pat; TOKAI-IRUD-1249 and upd(3)pat; TOKAI-IRUD-1180] with no diagnostic SNVs or CNVs. Thus, genetic diagnoses were obtained for 78 of 177 (44%) patients, and of these, 10 (13%) cases were diagnosed with diseases recognized after 2015, i.e., when this project was initiated. A considerable number of patients showed a milder phenotype (26 [33%]), a more severe phenotype (9 [12%]), or an atypical complex phenotype (17 [22%]) compared to conventional clinical presentation of the respective disease.

TOKAI-IRUD-1290 with upd(15)pat: The patient, a 2-year-old boy at the time of sample submission, was the third of three children of healthy non-consanguineous parents (Fig.2b). Gyrus dysplasia, suspected since the fetal period, was confirmed by magnetic resonance imaging (MRI) after birth (Fig.2a). He was tube fed due to difficulties with oral intake and a tracheostomy was performed after repeated aspiration pneumonia. He also had congenital hydronephrosis, congenital hypothyroidism, gastroesophageal reflux disease, developmental delay, epilepsy, deafness, and laryngotracheomalacia. ROH analysis identified a paternal UPD region over the entire length of the long arm of chromosome 15 [upd(15)pat], covering the region of the UBE3A gene, which led to a diagnosis of Angelman syndrome (OMIM#105830) (Fig.2b). Additionally, 11 homozygous rare variants were identified in a paternally derived UPD region, which included a DUOX2 (c.G1560C, p.E520D) variant. DUOX2 is a known causative gene for congenital hypothyroidism, but this particular variant has not been previously reported.

Clinical features and results of UPD analysis of TOKAI-IRUD-1290. (a) Brain MRI at the age of 2years showing cortical dysplasia of the temporal lobes (arrowheads) and corpus callosum dysgenesis (arrow). (b) Results of UPD analysis. A paternally inherited UPD region over the entire length of the long arm of chromosome 15 [upd(15)pat] was identified, which covers the region of the UBE3A gene. (c) H2O2-producing capacity of the DUOX2 proteins was measured with Amplex Red reagent in the presence of co-expressed DUOXA2-FLAG. The activity of the mutants were standardized based on those of the WT (100%) and mock-transfected control (0%). Data are representative of three independent experiments (each performed in triplicate) with similar results. T-bars indicate standard errors of the mean.*p<0 05 vs. WT (Welchs t-test). (d) Subcellular localization analysis using HA-tagged DUOX2 constructs (WT or E520D; green fluorescence). (e) Fluorescence immunostaining under permeabilized conditions revealed that the localization of E520D-DUOX2 was consistent with DUOXA2.

To verify the pathogenicity of the DUOX2 p.E520D missense substitution detected in this case, expression experiments were conducted using HEK293 cells wherein the H2O2-producing capacity of the E520D mutant in the presence of co-expressed DUOXA2-FLAG was evaluated. We show that the E520D mutant showed complete loss of H2O2-producing activity (Fig.2c). Visualization of subcellular localization using immunofluorescence revealed substantial differences in membrane expression levels between the WT and E520D mutant (Fig.2d,e), indicating that protein localization was affected by the missense substitution.

TOKAI-IRUD-1180 with upd(3)pat: This patient, a 3-year-old girl at the time of sample submission, was the only child of healthy non-consanguineous parents. She suffered seizures beginning on day 1 after birth and symptomatic epilepsy was suspected based on abnormalities detected on an electroencephalogram. However, the seizures ceased from day 14, when oral administration of phenobarbital was initiated. She was unable to sit and had poor language understanding at the time of sample submission. ROH analysis revealed a full-length UPD of chromosome 3 [upd(3)pat], and although 40 homozygous rare missense variants were identified on chromosome 3, it was not possible to arrive at a genetic diagnosis by WES analysis.

TOKAI-IRUD-1249 with upd(2)pat: The patient, a 4-month-old girl at the time of sample submission, was the only child of healthy non-consanguineous parents. A prenatal MRI confirmed hydrocephalus. She was born by scheduled cesarean section at gestational week 34 and suffered from deafness, bilateral club feet, bilateral hip dislocation, multiple joint contractures, congenital hydrocephalus, ventricular septal defect, developmental delay, short and mildly curved femurs, a bell-shaped rib cage, and a vagina without an external opening. ROH analysis revealed a full-length UPD of chromosome 2 [upd(2)pat]. She died of aspiration pneumonia at the age of 10months, and although 34 rare homozygous missense variants and one nonsense variant were identified on chromosome 2, WES analysis did not lead to a genetic diagnosis.

One pathogenic variant of a gene included in the ACMG recommendations for reporting incidental findings was detected in one patient (TOKAI-IRUD-1150), viz, c.C6952T in BRCA2. Additionally, discordant parentchild relationships were identified in three families.

Read more from the original source:
Whole-exome analysis of 177 pediatric patients with undiagnosed diseases | Scientific Reports - Nature.com

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