RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a ...limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.
Exome sequencing (ES) has become one of the important diagnostic tools in clinical genetics with a reported diagnostic rate of 25-58%. Many studies have illustrated the diagnostic and immediate ...clinical impact of ES. However, up to 75% of individuals remain undiagnosed and there is scarce evidence supporting clinical utility beyond a follow-up period of >1 year. This is a 3-year follow-up analysis to our previous publication by Mak et al. (
3:19, 2018), to evaluate the long-term clinical utility of ES and the diagnostic potential of exome reanalysis. The diagnostic yield of the initial study was 41% (43/104). Exome reanalysis in 46 undiagnosed individuals has achieved 12 new diagnoses. The additional yield compared with the initial analysis was at least 12% (increased from 41% to at least 53%). After a median follow-up period of 3.4 years, change in clinical management was observed in 72.2% of the individuals (26/36), leading to positive change in clinical outcome in four individuals (11%). There was a minimum healthcare cost saving of HKD$152,078 (USD$19,497; €17,282) annually for these four individuals. There were a total of six pregnancies from five families within the period. Prenatal diagnosis was performed in four pregnancies; one fetus was affected and resulted in termination. None of the parents underwent preimplantation genetic diagnosis. This 3-year follow-up study demonstrated the long-term clinical utility of ES at individual, familial and health system level, and the promising diagnostic potential of subsequent reanalysis. This highlights the benefits of implementing ES and regular reanalysis in the clinical setting.
Whole-exome sequencing (WES) has become an invaluable tool for genetic diagnosis in paediatrics. However, it has not been widely adopted in the prenatal setting. This study evaluated the use of WES ...in prenatal genetic diagnosis in fetuses with structural congenital anomalies (SCAs) detected on prenatal ultrasound.
Thirty-three families with fetal SCAs on prenatal ultrasonography and normal chromosomal microarray results were recruited. Genomic DNA was extracted from various fetal samples including amniotic fluid, chorionic villi, and placental tissue. Parental DNA was extracted from peripheral blood when available. We used WES to sequence the coding regions of parental-fetal trios and to identify the causal variants based on the ultrasonographic features of the fetus.
Pathogenic mutations were identified in three families (n = 3/33, 9.1%), including mutations in DNAH11, RAF1 and CHD7, which were associated with primary ciliary dyskinesia, Noonan syndrome, and CHARGE syndrome, respectively. In addition, variants of unknown significance (VUSs) were detected in six families (18.2%), in which genetic changes only partly explained prenatal features.
WES identified pathogenic mutations in 9.1% of fetuses with SCAs and normal chromosomal microarray results. Databases for fetal genotype-phenotype correlations and standardized guidelines for variant interpretation in prenatal diagnosis need to be established to facilitate the use of WES for routine testing in prenatal diagnosis.
Background
Autosomal recessive or compound heterozygous mutations in KLHL40 cause nemaline myopathy 8, which is one of the most severe forms of nemaline myopathy. The KLHL40 c.1516A>C variant has ...recently been reported as a founder mutation in southern Chinese.
Methods
We report six cases of nemaline myopathy 8 which involves the c.1516A>C variant, from five unrelated families of non‐consanguineous southern Chinese. The pre‐ and postnatal phenotypes of these cases were reviewed with emphasis on prenatal clinical features. Genetic testing for the founder mutation was performed on three patients with homozygous mutations.
Results
Common prenatal features included reduced fetal movement, polyhydramnios, breech presentation, and clubfeet. Two pregnancies were terminated. Four live‐born patients had postnatal features typical of nemaline myopathy 8. The length of survival ranged from 49 days to 17 months, with respiratory failure and infections being the principal causes of death. Haplotype analysis in three patients with homozygous mutation showed a shared haplotype block of 1.1727 cM spanning over the c.1516A>C variant, suggesting it is a southern Chinese‐specific founder mutation.
Conclusion
Analysis of the KLHL40 c.1516A>C variant should be considered in prenatal diagnosis of Chinese pregnant patients with suspected congenital neuromuscular disorders or with significant family history of congenital myopathies.
We reported six cases from five unrelated families of non‐consanguineous southern Chinese affected by nemaline myopathy 8, with either homozygous variants or compound heterozygous variants involving c.1516A>C in KLHL40. Pre‐ and postnatal phenotypes of the cases were reviewed, with emphasis on the prenatal clinical features.
Background In nonsyndromic conotruncal cardiac defects, the use of next-generation sequencing for clinical diagnosis is increasingly adopted, but gene-disease associations in research are only ...partially translated to diagnostic panels, suggesting a need for evidence-based consensus. Methods and Results In an exome data set of 245 patients with conotruncal cardiac defects, we performed burden analysis on a high-confidence congenital heart disease gene list (n=132) with rare (<0.01%) and ultrarare (absent in the Genome Aggregation Database) protein-altering variants. Overall, we confirmed an excess of rare variants compared with ethnicity-matched controls and identified 2 known genes (
) and 4 candidate genes supported by the literature (
, and
). Ultrarare variant analysis was performed in combination with 3 other published studies (n=1451) and identified 3 genes (
) to be significant, whereas a subgroup analysis involving 391 Chinese subjects identified only
as significant. Conclusions We suggest that these significant genes in our rare and ultrarare burden analyses warrant prioritization for clinical testing implied for rare inherited and de novo variants. Additionally, associations on ClinVar for these genes were predominantly variants of uncertain significance. Therefore, a more stringent assessment of gene-disease associations in a larger and ethnically diverse cohort is required to be prudent for future curation of conotruncal cardiac defect genes.
Rapid whole-exome sequencing (rWES) offers the potential for early diagnosis-predicated precision medicine. Previous evidence focused predominantly on infants from the intensive care unit (ICU). This ...study sought to examine the diagnostic and clinical utility, and the economic impact on clinical management of rWES in patients beyond infancy and ICU setting.
rWES was performed on a prospective cohort of patients with suspected monogenic disorder referred from territory-wide paediatric ICUs and non-ICUs in Hong Kong urging for rapid genetic diagnosis. All eligible families were invited. We aimed to achieve a rapid turnaround time (TAT) of 14 days. Clinical utility and costs associated with clinical management were assessed in diagnosed cases. Actual quantitative changes in healthcare utilisation were compared with a counterfactual diagnostic trajectory and/or with matched historical control whenever possible.
rWES were offered to 102 families and 32/102 (31%) patients received a molecular diagnosis, with a median TAT of 11 days. Clinical management changed in 28 of 32 diagnosed patients (88%), including but not limited to modifications in treatment, avoidance of surgeries, and informing decisions on redirection of care. Cost analysis was performed in eight patients. rWES was estimated to reduce hospital length of stay by 566 days and decrease healthcare costs by HKD$8,044,250 (GBP£796,460) for these eight patients. The net cost-savings after inclusion of rWES costs were estimated to be HKD$5,325,187 (GBP£527,246).
This study replicates the diagnostic capacity and rapid TAT of rWES in predominantly Chinese patients, and demonstrates diagnosis-predicated precision medicine and net healthcare savings. Findings were corroborated by evidence from multinational cohorts, combined as part of a meta-analysis. rWES merits consideration as a first-tier diagnostic tool for patients with urgent needs in the clinical setting.
Health and Medical Research Fund, HKU Seed Fund for Basic Research, The Society for the Relief of Disabled Children, and Edward and Yolanda Wong Fund.
Spinocerebellar ataxia 35 (SCA35) has been associated with pathogenic mutations in the gene TGM6. In a Chinese exome sequencing cohort, we identified 8 families with reported TGM6 variants sharing no ...features of SCA35. Considering this finding, we reviewed the public database gnomAD and found these variants to be significantly more common in the East Asians than in other ethnic groups (P < 0.0001). Gene constraint metrics showed that both missense and loss-of-function variants in TGM6 are likely to be tolerated and there is no regional constraint. By performing inflation analysis, it demonstrated that the cumulative frequency of TGM6 reported pathogenic variants is at least 111-fold inflated over disease prevalence of all autosomal dominant SCAs, indicating a high chance of misdiagnosis or low penetrance. Misclassification of benign or low penetrant variants as pathogenic is a significant problem that often results in genetic misdiagnosis. This highlights the necessity of evaluating variant pathogenicity with sequencing of genomes from diverse populations, both from asymptomatic controls and phenotypically different patients, in order to ensure accurate classification of variants.
•TGM6 mutations are previously associated with spinocerebellar ataxia (SCA) type 35.•We identified 8 families with reported TGM6 variants showing no features of SCA35.•GnomAD data suggest that TGM6 variants are tolerated and show no regional constraint.•Frequency of TGM6 variant carrier is at least 111-fold above the prevalence of SCA.•With the above observation, we question the role of TGM6 in the pathogenesis of SCA35.
Background
Neuromuscular disorders (NMDs) comprise a group of heterogeneous genetic diseases with a broad spectrum of overlapping the clinical presentations that makes diagnosis challenging. Notably, ...the recent introduction of whole‐exome sequencing (WES) is introducing rapid changes on the genetic diagnosis of NMDs. We aimed to investigate the diagnostic value of WES for pediatric‐onset NMDs.
Methods
We applied integrated diagnostic approach and performed WES in 50 Chinese subjects (30 males, 20 females) with undiagnosed pediatric‐onset NMDs despite previous specific tests. The patients were categorized in four subgroups according to phenotyping and investigation findings. Variants on NMDs gene list and open exome analysis for those with initial negative findings were identified.
Results
WES identified causative variants in ACTA1 (n = 2), POMT1, COL6A1 (n = 2), MTMR2, LMNA, SELENON, DNM2, TGFB1, MPZ, IGHMBP2, and LAMA2 in 13 patients. Two subjects have variants of uncertain significance (VUSs) in TTN and SCN11A, unlikely to be pathogenic due to incompatible phenotypes. The mean interval time from symptom onset to genetic diagnosis was 10.4 years (range from 1 month to 33 years). The overall diagnostic yield of WES in our cohort was 26%. Open exome analysis was necessary to identify the pathogenic variant in TGFB1 that caused skeletal dysplasia with neuromuscular presentation.
Conclusion
Our study shows a clear role of WES in the pathway of integrated diagnostic approach to shorten the diagnostic odyssey in patients with rare NMDs.
Our study achieved a diagnostic yield from WES of 26% (13/50). These cases were diagnostically challenging as prior investigations failed to give clues on specific genetic diagnosis. Our findings are compatible to previous studies observing the diagnostic yield tends to be lower in cohorts that have already undergone prior extensive evaluations.
Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of ...protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.
•Two Chinese children with same novel CHKB mutation have different presentation.•MDCMC, a rare multi-system disease, can have early mortality from cardiomyopathy.•MDCMC has selective muscle pattern ...involvement on MRI lower limb muscles.
Megaconial congenital muscular dystrophy (CMD) is a rare form of congenital muscular dystrophy attributed to an autosomal recessive CHKB mutation. We report two unrelated Chinese girls with Megaconial CMD who harbored the same novel homozygous CHKB mutation but exhibited different phenotypes. Patient 1, who is now 8 years old, has autism, intellectual disabilities, mild girdle weakness, and characteristic muscle biopsy with COX-negative fibers. Patient 2, now 12 years old, has limited intelligence and marked weakness, with scoliosis, hip subluxation and early loss of ambulation. Both exhibited mildly elevated creatine kinase levels, have relative sparing of adductor longus and extensor digitorum longus on MRI leg muscles, and a c.598del (p.Gln200Argfs*11) homozygous CHKB loss-of-function mutation. Their parents are heterozygous carriers. This is the first report of Megaconial CMD in Chinese patients demonstrating the pathogenicity of the identified homozygous CHKB variant. A case review of all previously reported patients of different ethnicities is also included.
PDF
