Rapid exome sequencing as the first-tier investigation for diagnosis of acutely and severely ill children and adults in Thailand.

The use of rapid DNA sequencing technology in severely ill children in developed countries can accurately identify diagnoses and positively impact patient outcomes. This study sought to evaluate the outcome of Thai children and adults with unknown etiologies of critical illnesses with the deployment of rapid whole exome sequencing (rWES) in Thailand. We recruited 54 unrelated patients from 11 hospitals throughout Thailand. The median age was 3 months (range, 2 days-55 years) including 47 children and 7 adults with 52% males. The median time from obtaining blood samples to issuing the rWES report was 12 days (range, 5-27 days). A molecular diagnosis was established in 25 patients (46%), resulting in a change in clinical management for 24 patients (44%) resulting in improved clinical outcomes in 16 patients (30%). Four out of seven adult patients (57%) received the molecular diagnosis which led to a change in management. The 25 diagnoses comprised 23 different diseases. Of the 34 identified variants, 15 had never been previously reported. This study suggests that use of rWES as a first-tier investigation tool can provide tremendous benefits in critically ill patients with unknown etiology across age groups in Thailand.

Nine patients with KCNQ2-related neonatal seizures and functional studies of two missense variants.

Mutations in KCNQ2 encoding for voltage-gated K channel subunits underlying the neuronal M-current have been associated with infantile-onset epileptic disorders. The clinical spectrum ranges from self-limited neonatal seizures to epileptic encephalopathy and delayed development. Mutations in KCNQ2 could be either gain- or loss-of-function which require different therapeutic approaches. To better understand genotype-phenotype correlation, more reports of patients and their mutations with elucidated molecular mechanism are needed. We studied 104 patients with infantile-onset pharmacoresistant epilepsy who underwent exome or genome sequencing. Nine patients with neonatal-onset seizures from unrelated families were found to harbor pathogenic or likely pathogenic variants in the KCNQ2 gene. The p.(N258K) was recently reported, and p. (G279D) has never been previously reported. Functional effect of p.(N258K) and p.(G279D) has never been previously studied. The cellular localization study demonstrated that the surface membrane expression of Kv7.2 carrying either variant was decreased. Whole-cell patch-clamp analyses revealed that both variants significantly impaired Kv7.2 M-current amplitude and density, conductance depolarizing shift in voltage dependence of activation, membrane resistance, and membrane time constant (Tau), indicating a loss-of-function in both the homotetrameric and heterotetrameric with Kv7.3 channels. In addition, both variants exerted dominant-negative effects in heterotetrameric with Kv7.3 channels. This study expands the mutational spectrum of KCNQ2- related epilepsy and their functional consequences provide insights into their pathomechanism.

Severe coarctation of the aorta, developmental delay, and multiple dysmorphic features in a child with SMAD6 and SMARCA4 variants.

Pathogenic variants in SMARCA4 cause Coffin-Siris syndrome (CSS) while those in SMAD6 lead to aortic valve disease and other dysmorphisms. We identified a 6-year-old Thai boy with features of CSS alongside unusual manifestations including, very severe coarctation of the aorta (CoA) requiring coarctectomy in the neonatal period and bilateral radioulnar synostoses. Trio exome sequencing revealed that the patient harbored two de novo variants, a missense c.2475G > T, p.(Trp825Cys) in SMARCA4 and a nonsense c.652C > T, p.(Gln218Ter) in SMAD6. Both of which have never been previously reported. The clinical presentations in our patient are a result of the combinational features of each genetic variant: the SMARCA4 p.(Trp825Cys) variant leads to facial features of Coffin Siris syndrome and Dandy-Walker malformation, while the SMAD6 p.(Gln218Ter) variant underlies radioulnar synostosis. Interestingly, the severity of CoA in the proband is beyond the phenotypic spectra of each genetic variant and may be a result of the synergistic effects of both variants. Here, we report a child with variants in SMARCA4 or SMAD6 with combined features of each plus a severe CoA, possibly due to an additive effect of each variant.

Long-read Amplicon Sequencing of the CYP21A2 in 48 Thai Patients With Steroid 21-Hydroxylase Deficiency.

CONTEXT: Congenital adrenal hyperplasia is most commonly caused by 21-hydroxylase deficiency (21-OHD), an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in CYP21A2. With a highly homologous pseudogene and various types of single nucleotide and complex structural variants, identification of PVs in CYP21A2 has been challenging. OBJECTIVE: To leverage long-read next-generation sequencing combined with locus-specific polymerase chain reaction (PCR) to detect PVs in CYP21A2 and to determine its diagnostic yield in patients with 21-OHD. METHODS: Forty-eight Thai patients with 21-OHD comprising 38 sporadic cases and 5 pairs of siblings were enrolled. Two previously described locus-specific PCR methods were performed. Amplicons were subject to long-read sequencing. RESULTS: Ninety-six PVs in CYP21A2 in the 48 patients were successfully identified. The combined techniques were able to detect 26 structural chimeric variants (27%; 26/96) in 22 patients with 18 having monoallelic and 4 having biallelic chimeras. The remaining PVs were pseudogene-derived mutations (63%; 60/96), entire gene deletions (2%; 2/96), missense variants (3%; 3/96), a splice-site variant (2%; 2/96), frameshift variants (2%; 2/96), and a nonsense variant (1%; 1/96). Notably, a splice-site variant, IVS7 + 1G > T, which was identified in a pair of siblings, has not previously been reported. CONCLUSIONS: Our approach exploiting locus-specific PCR and long-read DNA sequencing has a 100% diagnostic yield for our cohort of 48 patients with 21-OHD.