As a tertiary genetic counseling and prenatal diagnosis center, our center served 290 families with individuals suspected of rare monogenic diseases during the 4-year study, and 142 (nearly 50%) of those patients had GDD/ID. After excluding 13 patients for missing information, 18 patients for uncertain diagnosis and 3 patients who had pathogenic variants along with atypical manifestations that could not be explained by the variants, we included a total of 108 subjects at preliminary screening. Further reviewing the case history, we excluded 16 patients who reached developmental lime stones normally in early stage and suffered GDD secondary to developmental regression, 5 patients that experienced abundant epileptiform activity in the neonatal period and then showed developmental arrest or regression and 7 patients for early death. Finally, we considered a total of 81 subjects and their core family members. (Figure S1).
- Demographic features and diagnostic courses
The 81 subjects came from17 out of the 31 provinces and municipalities in mainland China. The numbers of cases inherited in AR, AD, and XL patterns were 30 (37.0%), 38 (47.0%) and 13 (16.0%), respectively. The median age was 50 months (IQR, 25-76.5), and 51 (63.0%) participants were male. The median age of onset was 3.5 months (IQR, 3-7), ranging from the day of birth to 2 years and 3 months. And 70 (95%) participants had symptoms before 1 year, all participants presented GDD before 3 years of age.
The median interval from disease onset to genetic diagnosis was 21 months (IQR, 9-55.5m), ranging from 1 month to 12 years, and the median duration from genetic diagnosis to genetic counseling was 10 months (IQR, 4-23m; range, 0-6.3y). The median number of hospital referrals was 4 (IQR, 2-5; range, 2-8) (Table 1 and Supplementary table 1).
Among the 81 subjects, 32 patients were diagnosed as neurodevelopmental disorders (30 syndromic ID and 2 non-syndromic ID), 20 patients had metabolic disorders, 17 patients were genetic epilepsy, and 7 patients had other neurogenetic disorders (3 neuromuscular disorders, 1 developmental brain disorder, 1 genodermatosis, and 1 multiple congenital anormaly). In addition, 54 patients had static courses (GDD had slowly improvement), 13 patients presented progressive courses (GDD followed by psychomotor regression/arrest), and 14 patients of unknown courses. The results were listed in Figure1, Table 2 and supplementary 2 in detail.
Since developmental scale should be evaluated after 3 months old, GDD might not be their first manifestations. Nearly 25% (22/81) of patients had abnormalities on appearance such as microcephalus, macrocephalus, facial anomalies, short statue, abnormal skin, hair and iris, and kyphoscoliosis, before GDD. Besides 6 patients with development and epileptic encephalopathy presented epilepsy before GDD.
Of the 81 subjects, 15 (22.4%) patients had mild-moderate GDD/ID, and the other 52 (77.6%) had severe to profound GDD/ID. The percentages of patients diagnosed by WES were 47% (7/15) in mild-moderate subjects and 60% (30/50) in severe-profound subjects , the difference of diagnostic methods used in mild and severe patients did not reach statistical significance (Table 5).
Epilepsies were co-existing in 58%(47/81) of patients. Among them, 7 patients had focal epilepsies, 16 had generalized epilepsies, 14 had combined generalized and focal epilepsies and 10 patients were classified as unknown type. Autism spectrum disorders (ASD) were confirmed in 5 patients and 3 patients had borderline ASD.
Other common presentations including facial anomalies (14 [17.2%]), microcephaly (17 [20.9%]), macrocephaly (7 [8.5%]), vision impairment (10 [12.8%]) and hearing loss (4 [5.0%]). Twelve of 60 (24%) patients had low weight, and 5/48 (10%) had short stature. Organ involvements were also observed in 16%(13/81) of subjects: 5 (6%) patients had heart involvements, 3 (4%) had liver involvements, 1 (1%) had kidney involvement, 3 (3.9%) had abnormal skin or hair manifestations, 2 had bone anomalies and 1 patient with thyroid hormone abnormality (Table 2 and 7).
In addition, 48 patients had abnormal brain imaging, the primary abnormalities including cerebral white matter changes (28 [53%]), hypoplasia of corpus callosum (12 [25%]) and cerebellar abnormalities (7 [14.6%]) (Table 6).
The majority of affected individuals (94%, 75/80) were simplex cases (a single occurrence in a family), and only 5 (6%) patients had a positive family history.
Notably, 12.5% (10/80) of patients had abnormal prenatal ultrasound findings. (The details are listed in table 8). In addition, 4 patients experienced hypoxic events during labor, the impact of hypoxic events on their development had been excluded by pediatric neurologists. .
- Genetic characteristics
In total, 111 pathogenic/likely pathogenic variants were found in 62 different genes among the 81 pedigrees. Of these genes, 28 genes were transmitted in the AR pattern, 25 in the AD pattern and 9 in the XL pattern. In order to analyze the disparity in genetic spectrum between different inherited models, repeated variants were included in the calculation. The results are presented in Table 3 and Supplement 3 in detail. Among these disease-causing variants, there were 51 (45.9%) missense variants, 23 (20.7%) nonsense variants, 24 (21.6%) frameshift variants, 4 (3.6%) small deletion variants, 9 (8.1%) variants that caused splicing defects.
Gene ontology accumulation analyses indicated that those genes took part in multiple biological processes, including nervous system development, nervous impulse transmission, ion transport and metabolism. Genes associated with ion channel transport and nervous system development were mainly inherited in the AD model, while genes related to metabolism were mainly transmitted in AR or XL patterns (Supplementary Figure 2).
Among the 62 different causative genes, SCN2A, SHANK3 and STXBP1 were found in 3 patients each; and ALG1, CHD2, FOXG1, GATAD2B, GFAP, GLB1, GRIN2B, IDS, KCNQ2, PAFAH1B1, PCDH19, SLC9A6 and SYNGAP1 in 2 patients each. The other 46 out of 62 genes were observed to have pathogenic variants only once each in this cohort.
Most variants were unique in this cohort, while two variants were relatively common. One was the c.1343 A>T in the GLB1 gene, which occurred in 3 alleles of 2 patients (patient 42/44) among 2 patients with GLB1-related diseases.Iit was a high frequency variant in GLB1 . The other was a de novo variant c.235C>T in GFAP, which was detected in two unrelated patients (Nos.36 and 37) with Alexander Disease. It was a variant that had been reported several times[35-37] but absent in the Normal Population Database (GnomAD and 1000G). Additionally, a homozygous substitution variant, c.1510C>T, was found in patients (Nos. 51) with Tay-Sachs disease, confirmed by hexosaminidase A enzyme deficiency (<1.1nmol/mg/h). Multiple studies[38-40] have reported the pathogenicity of these variants, suggesting that the 1510th base pair in the coding sequence of HEXA (NM_000520) was a common variant position.
Notably, 56 (50.4%) variants were identified as novel variants, and 54 (48.6%) variants have been included in disease databases (ClinVar or HGMD) or reported in PubMed articles. The rate was similar to that in previous studies[41-45]. The proportions of novel variants in ARID, ADID and XLID were 43.8%, 65% and 50%, respectively. This suggests that variant spectrum in known ID genes have not been fully explored in all inheritance patterns. The higher rate of novel variants in ADID might be explained by the fact that most variants arose de novo in the AD pattern.
The major difference among ARID, ADID and XLID lies in the origin of variants. Of the 30 patients with ARID, 27 (90%) patients carried compound heterozygous variants, and 3 (10%) patients harbored homozygous variants. We confirmed that in all patients, the two abnormal alleles were separately inherited from healthy outbred parents who carried the heterozygous variants. Among 38 patients with ADID, 37 (97.4%) variants arose de novo, 1 variant was transmitted from a mosaic father. Of the 13 patients with XLID, 7 (53.8%) patients (2 males, 5 female) had de novo variants, 5 male patients harbored hemizygous variants inherited from their asymptomatic heterozygous mother, and 1 female (patient 70) inherited the heterozygous variant c.445C>T in PCDH19 from her non-symptomatic father. This unique characteristic was supported by previous reports .
In addition, parental somatic mosaicism was found in 2 cases. One is patient 33, who presented with facial dysmorphism and GDD, had a c.941del in GATAD2B. The variant was also detected at a low frequency in his paternal peripheral blood genomic DNA but absent in samples of his healthy mother and sister. Therefore, it is likely that the father carries somatic and germline mosaicism for this variant. The other is patient 93, who harbored a hemizygous c.1153C>T in SLC9A6. And his mother was suspected to have the variant in mosaic state with a low peak in her peripheral blood Sanger sequencing.
- Prenatal diagnostic results
By the time of follow-up, totally 33 families underwent prenatal tests to determine whether the next child would harbor the same pathogenic variants as the index patient in the fetal period. As demonstrated in Table 4 and Supplement 4, among them, 15 cases were ARID, 13 cases were ADID and 4 were XLID. And 28 (84.8%) patients chose amniocentesis, and 5 (15.2%) patients underwent chorionic villus sampling. Among the 15 AR cases, 4 fetuses were found to carry two pathogenic variants that originated from parents who were healthy carriers, 9 fetuses harbored one variant, and 2 fetuses did not have any variants. Among the 13 AD cases, 11 fetuses did not have the variants, while 2 fetuses carried the same variants as the proband in the GATAD2B gene. Of the 5 XL cases, only 1 fetus harbored the pathogenic variant. The recurrence rates of AR, AD and XL modes were 26.7%, 15.4% and 20% respectively. All variants carried by fetuses were verified after birth or induction of labor.