Expansion of the clinical and molecular spectrum of WWOX-related epileptic encephalopathy.

WWOX biallelic loss-of-function pathogenic single nucleotide variants (SNVs) and copy number variants (CNVs) including exonic deletions and duplications cause WWOX-related epileptic encephalopathy (WOREE) syndrome. This disorder is characterized by refractory epilepsy, axial hypotonia, peripheral hypertonia, progressive microcephaly, and premature death. Here we report five patients with WWOX biallelic predicted null variants identified by exome sequencing (ES), genome sequencing (GS), and/or chromosomal microarray analysis (CMA). SNVs and intragenic deletions of one or more exons were commonly reported in WOREE syndrome patients which made the genetic diagnosis challenging and required a combination of different diagnostic technologies. These patients presented with severe, developmental and epileptic encephalopathy (DEE), and other cardinal features consistent with WOREE syndrome. This report expands the clinical phenotype associated with this condition, including failure to thrive in most patients and epilepsy that responded to a ketogenic diet in three patients. Dysmorphic features and abnormal prenatal findings were not commonly observed. Additionally, recurrent pancreatitis and sensorineural hearing loss each were observed in single patients. In summary, these phenotypic features broaden the clinical spectrum of WOREE syndrome.

Low-pass genome sequencing: a validated method in clinical cytogenetics.

Clinically significant copy-number variants (CNVs) known to cause human diseases are routinely detected by chromosomal microarray analysis (CMA). Recently, genome sequencing (GS) has been introduced for CNV analysis; however, sequencing depth (determined by sequencing read-length and read-amount) is a variable parameter across different laboratories. Variating sequencing depths affect the CNV detection resolution and also make it difficult for cross-laboratory referencing or comparison. In this study, by using data from 50 samples with high read-depth GS (30×) and the reported clinically significant CNVs, we first demonstrated the optimal read-amount and the most cost-effective read-length for CNV analysis to be 15 million reads and single-end 50 bp (equivalent to a read-depth of 0.25-fold), respectively. In addition, we showed that CNVs at mosaic levels as low as 30% are readily detected, furthermore, CNVs larger than 2.5 Mb are also detectable at mosaic levels as low as 20%. Herein, by conducting a retrospective back-to-back comparison study of low-pass GS versus routine CMA for 532 prenatal, miscarriage, and postnatal cases, the overall diagnostic yield was 22.4% (119/532) for CMA and 23.1% (123/532) for low-pass GS. Thus, the overall relative improvement of the diagnostic yield by low-pass GS versus CMA was ~ 3.4% (4/119). Identification of cryptic and clinically significant CNVs among prenatal, miscarriage, and postnatal cases demonstrated that CNV detection at higher resolutions is warranted for clinical diagnosis regardless of referral indications. Overall, our study supports low-pass GS as the first-tier genetic test for molecular cytogenetic testing.

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans.

Mitochondrial diseases in Hong Kong: prevalence, clinical characteristics and genetic landscape.

OBJECTIVE: To determine the prevalence of mitochondrial diseases (MD) in Hong Kong (HK) and to evaluate the clinical characteristics and genetic landscape of MD patients in the region. METHODS: This study retrospectively reviewed the phenotypic and molecular characteristics of MD patients from participating public hospitals in HK between January 1985 to October 2020. Molecularly and/or enzymatically confirmed MD cases of any age were recruited via the Clinical Analysis and Reporting System (CDARS) using relevant keywords and/or International Classification of Disease (ICD) codes under the HK Hospital Authority or through the personal recollection of treating clinicians among the investigators. RESULTS: A total of 119 MD patients were recruited and analyzed in the study. The point prevalence of MD in HK was 1.02 in 100,000 people (95% confidence interval 0.81-1.28 in 100,000). 110 patients had molecularly proven MD and the other nine were diagnosed by OXPHOS enzymology analysis or mitochondrial DNA depletion analysis with unknown molecular basis. Pathogenic variants in the mitochondrial genome (72 patients) were more prevalent than those in the nuclear genome (38 patients) in our cohort. The most commonly involved organ system at disease onset was the neurological system, in which developmental delay, seizures or epilepsy, and stroke-like episodes were the most frequently reported presentations. The mortality rate in our cohort was 37%. CONCLUSION: This study is a territory-wide overview of the clinical and genetic characteristics of MD patients in a Chinese population, providing the first available prevalence rate of MD in Hong Kong. The findings of this study aim to facilitate future in-depth evaluation of MD and lay the foundation to establish a local MD registry.

Case Report: Novel Biallelic Variants in the COL18A1 Gene in a Chinese Family With Knobloch Syndrome.

Knobloch syndrome is a rare collagenopathy characterized by severe early onset myopia, retinal detachment, and occipital encephalocele with various additional manifestations due to biallelic changes in the COL18A1 gene. Here we reported a Chinese family with two affected siblings presented with antenatal occipital encephalocele, infantile onset retinal detachment, and pronounced high myopia at early childhood. Quartet whole exome sequencing was performed in this family and identified that both siblings carried novel compound heterozygous variants in the COL18A1 gene (NM_001379500.1): the maternally inherited variant c.1222-1G>A at the consensus acceptor splice site of intron 8, and the paternally inherited frameshift variant c.3931_3932delinsT p.(Gly1311Serfs*25) in the last exon. Both patients had successful surgical treatment for the occipital encephalocele soon after birth. They had normal neurocognitive outcome and good general conditions examined at the age of 7 years old for the elder sister and 4 years old for the younger brother. The younger brother developed infantile onset retinal detachment at 7 months of age while the sister had high myopia without signs of retinal detachment until 7 years old. This report expands the phenotype and genotype spectrum of Knobloch syndrome with antenatal and postnatal findings.

Implementation of Public Funded Genome Sequencing in Evaluation of Fetal Structural Anomalies.

With the advancements in prenatal diagnostics, genome sequencing is now incorporated into clinical use to maximize the diagnostic yield following uninformative conventional tests (karyotype and chromosomal microarray analysis). Hong Kong started publicly funded prenatal genomic sequencing as a sequential test in the investigation of fetal structural anomalies in April 2021. The objective of the study was to evaluate the clinical performance and usefulness of this new service over one year. We established a web-based multidisciplinary team to facilitate case selection among the expert members. We retrospectively analyzed the fetal phenotypes, test results, turnaround time and clinical impact in the first 15 whole exome sequencing and 14 whole genome sequencing. Overall, the molecular diagnostic rate was 37.9% (11/29). De novo autosomal dominant disorders accounted for 72.7% (8/11), inherited autosomal recessive disorders for 18.2% (2/11), and inherited X-linked disorders for 9.1% (1/11). The median turnaround time for ongoing pregnancy was 19.5 days (range, 13-31 days). Our study showed an overall clinical impact of 55.2% (16/29), which influenced reproductive decision-making in four cases, guided perinatal management in two cases and helped future family planning in ten cases. In conclusion, our findings support the important role of genome sequencing services in the prenatal diagnosis of fetal structural anomalies in a population setting. It is important to adopt a multidisciplinary team approach to support the comprehensive genetic service.

Neonatal epidermolysis bullosa: lessons to learn about genetic counseling.

BACKGROUND: Epidermolysis Bullosa (EB) is a heterogeneous group of congenital blistering diseases that usually presents in the neonatal period. EB is classified into three major categories, each with many subtypes based on the precise location at which separation or blistering occurs, namely epidermolysis bullosa simplex (EBS), junctional epidermolysis bullosa (JEB) and dystrophic epidermolysis bullosa (DEB). METHODS: We describe genetics of neonatal EB in Hong Kong. RESULTS: Two neonates of consanguineous Pakistani parents had the EB-Pyloric Atresia (EB-PA) variant. One had a 4 kb homozygous deletion of exon 19-25 of the ITGB4 gene, and the other with only a histopathological diagnosis. Both died of sepsis in infancy. Aberrant COL7A1 mutations in the dominant and recessive EB were described. Genetic analysis, together with histopathological classification is important to aid prognosis and counseling. JEB and EB-PA are associated with consanguinity and mortality during infancy. Morbidity and prognosis of the autosomal dominant DEB are optimistic. The autosomal recessive DEB is more severe, with neonatal onset and recurrent blistering. It is also associated with chronicity and malignant changes when the child reaches adulthood. CONCLUSION: Exact genetic diagnosis aids in counseling of the family concerning the prognosis in the affected child and the risk of affected children in future pregnancies.

Clinical Implementation of Expanded Carrier Screening in Pregnant Women at Early Gestational Weeks: A Chinese Cohort Study.

Demands for expanded carrier screening (ECS) are growing and ECS is becoming an important part of obstetrics practice and reproductive planning. The aim of this study is to evaluate the feasibility of a small-size ECS panel in clinical implementation and investigate Chinese couples' attitudes towards ECS. An ECS panel containing 11 recessive conditions was offered to Chinese pregnant women below 16 gestational weeks. Sequential testing of their partners was recommended for women with a positive carrier status. The reproductive decision and pregnancy outcome were surveyed for at-risk couples. A total of 1321 women performed ECS successfully and the overall carrier rate was 19.23%. The estimated at-risk couple rate was 0.83%. Sequential testing was performed in less than half of male partners. Eight at-risk couples were identified and four of them performed prenatal diagnosis. Our study demonstrated that a small-size ECS panel could yield comparable clinical value to a larger-size panel when the carrier rate of the individual condition is equal or greater than 1%. In addition, more than half of male partners whose wives were carriers declined any types of sequential testing possibly due to a lack of awareness and knowledge of genetic disorders. Genetic education is warranted for the better implementation of ECS.

Severe Generalized Epidermolysis Bullosa Simplex in Two Hong Kong Children due to De Novo Variants in KRT14 and KRT5.

We report two Hong Kong children with severe generalized epidermolysis bullosa simplex (EBS), the most severe form of EBS, without a family history of EBS. EBS is a rare genodermatosis usually inherited in an autosomal dominant fashion although rare autosomal recessive cases have been reported. Genetic studies in these patients showed that the first case was due to a novel de novo heterozygous variant, c.377T>G (NM_000526.5 (c.377T>G, p.Leu126Arg)) in the KRT14 gene and the second case was due to a rare de novo heterozygous variant c.527A>G (NM_000424.4, c.527A>G, p.Asn176Ser) in the KRT5 gene. To our knowledge, the c.377T>G variant in the KRT14 gene has not been previously reported, and the c.527A>G variant in the KRT5 gene is a rare cause of severe generalized EBS. In severe generalized EBS, infants exhibit severe symptoms at the onset; however, they tend to improve with time. A precise genetic diagnosis in these two cases aided in counseling the families concerning the prognosis in their affected children and the recurrence risk for future pregnancies.

Primary coenzyme Q10 deficiency-7: expanded phenotypic spectrum and a founder mutation in southern Chinese.

Primary coenzyme Q10 deficiency-7 (COQ10D7) is a rare mitochondrial disease caused by biallelic mutations in COQ4. Here we report the largest cohort of COQ10D7 to date, with 11 southern Chinese patients confirmed with biallelic COQ4 mutations. Five of them have the classical neonatal-onset encephalo-cardiomyopathy, while the others have infantile onset with more heterogeneous clinical presentations. We also identify a founder mutation COQ4 (NM_016035.5): c.370G>A, p.(Gly124Ser) for COQ10D7, suggesting a higher chance of occurrence in the southern Chinese. This study helps improve understanding of the clinical spectrum of this disorder.