Clinical and molecular characteristics of hemophilia A affected individuals and carriers: A 24 years experience from three centers.

Hemophilia A is a rare bleeding disorder with variable expressivity and allelic heterogeneity. Despite the advancement of prenatal diagnostics and molecular studies, the number of studies reviewing the reproductive choices of hemophilia A carriers and affected individuals remains limited. Through this retrospective review, we hope to gain a deeper understanding of hemophilia A-affected individuals' clinical and molecular characteristics, as well as the reproductive choices of the at-risk couples. A total of 122 individuals harboring likely causative F8 gene alterations from 64 apparently unrelated families attending three centers between 3/2000 and 3/2023 were included in this study. Their clinical and molecular findings as well as reproductive choices were gathered in a clinical setting and verified through the electronic medical record database of the public health system. Forty-seven affected males and 75 female heterozygous carriers were included in the analysis. Among 64 apparently unrelated families, 36 distinct pathogenic/likely pathogenic variants were identified, of which 30.6% (11/36) of variants were novel. While the majority of clinical findings and genotype-phenotype correlations appear to be in accordance with existing literature, female carriers who had no fertility intention were significantly more likely to have affected sons than those who had fertility intention (5/19 vs. 4/5; p = 0.047). Through this retrospective review, we summarized the clinical and molecular characteristics of 122 individuals harboring pathogenic/likely pathogenic F8 variants, as well as their fertility intentions and reproductive outcomes. Further studies are required to look into the considerations involved in reproductive decision-making.

A Baby With Complete Androgen Insensitivity Syndrome and the Fortuitous Discovery of 45,X/46,XY Mosaicism.

Disorders of sex development (DSD) are caused by defects in the complex sexual differentiation cascade, resulting in discordance among an individual's genetic, gonadal, and genital sexes. It affects one in 4,500 live births. A wide spectrum of genital phenotypes can be found depending on the underlying pathogenic mechanism and the developmental stage that is affected. We herein report a newborn with female external genitalia but palpable gonads at labia majora with normal testicular function and structure, which is typical of complete androgen insensitivity syndrome (CAIS). The genetic study revealed 45,X/46,XY mosaicism and c.2081A>C missense androgen receptor gene mutation, indicating the likelihood of co-existing CAIS. This case demonstrated the importance of correlating genital phenotype and the underlying pathogenic mechanism, to provide appropriate management of DSD. Important considerations on managing the gonads about the risks of gonadal malignancies are also discussed.

Prenatal diagnosis of Myhre syndrome with a heterozygous pathogenic variant in SMAD4 gene presented with thick nuchal translucency and cardiac abnormalities.

Prenatal testing was performed in a 39-year-old Chinese pregnant woman referred for increased nuchal translucency measuring 5.7 mm. Non-invasive prenatal testing and SNP array study on amniotic fluid samples were normal. Whole exome sequencing (WES) was initiated further as the fetus had pericardial effusion of 1.2 mm, thickened myocardium over the right ventricular lateral wall and aberrant right subclavian artery. A detailed fetal echocardiogram also revealed persistent left superior vena cava and dilated coronary sinus at 20 weeks. From whole exome sequencing of the trio, a de novo heterozygous variant NM_005359.5(SMAD4): c.1499T>C (p.Ile500Thr) was detected. This pathogenic variant has been reported in the postnatal case cohort of Myhre syndrome. This condition is characterized by facial dysmorphism, intellectual disability, hearing loss, skeletal abnormalities and potential life threatening respiratory or cardiovascular manifestations. Termination of pregnancy was performed at 23 weeks. Small chins, pre-axial polydactyly, brachydactyly and clinodactyly were noted in the abortus. Ultrasound findings of increased nuchal translucency, thickened myocardium and pericardial effusion prompted further genetic evaluation for the prenatal diagnosis of Myhre syndrome by whole exome sequencing.

Public and Healthcare Provider Receptivity toward the Retention of Dried Blood Spot Cards and Their Usage for Extended Genetic Testing in Hong Kong.

Dried blood spot (DBS) cards from newborn screening (NBS) programs represent a wealth of biological data. They can be stored easily for a long time, have the potential to support medical and public health research, and have secondary usages such as quality assurance and forensics, making it the ideal candidate for bio-banking. However, worldwide policies vary with regard to the duration of storage of DBS cards and how it can be used. Recent advances in genomics have also made it possible to perform extended genetic testing on DBS cards in the newborn period to diagnose both actionable and non-actionable childhood and adult diseases. Both storage and secondary uses of DBS cards raise many ethical, clinical, and social questions. The openness of the key stakeholders, namely, parents and healthcare providers (HCPs), to store the DBS cards, and for what duration and purposes, and to extended genetic testing is largely dependent on local cultural-social-specific factors. The study objective is to assess the parents' and HCPs' awareness and receptivity toward DBS retention, its secondary usage, and extended genetic testing. A cross-sectional, self-administrated survey was adopted at three hospitals, out of which two were public hospitals with maternity services, between June and December 2022. In total, 452 parents and 107 HCPs completed and returned the survey. Overall, both HCPs and parents were largely knowledgeable about the potential benefits of DBS card storage for a prolonged period and its secondary uses, and they supported extended genetic testing. Knowledge gaps were found in respondents with a lower education level who did not know that a DBS card could be stored for an extended period (p < 0.001), could support scientific research (p = 0.033), and could aid public health research, and future policy implementation (p = 0.030). Main concerns with regard to DBS card storage related to potential privacy breaches and anonymity (Parents 70%, HCPs 60%). More parents, compared to HCPs, believed that storing DBS cards for secondary research does not lead to a reciprocal benefit to the child (p < 0.005). Regarding extended genetic testing, both groups were receptive and wanted to know about actionable childhood- and adult-onset diseases. More parents (four-fifths) rather than HCPs (three-fifths) were interested in learning about a variant with unknown significance (p < 0.001). Our findings report positive support from both parents and HCPs toward the extended retention of DBS cards for secondary usage and for extended genetic testing. However, more efforts to raise awareness need to be undertaken in addition to addressing the ethical concerns of both parents and HCPs to pave the way forward toward policy-making for DBS bio-banking and extended genetic testing in Hong Kong.

Fibrodysplasia ossificans progressiva in Hong Kong-A case report series.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare condition. The diagnosis could be challenging due to its rarity and non-specific presenting symptoms. However, early diagnosis and appropriate management help in preserving patients' function and quality of life. Herein, we report the diagnostic journeys and clinical courses of 8 patients with FOP in Hong Kong and illustrate the challenges involved.

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.

KCNQ2 Encephalopathy and Responsiveness to Pyridoxal-5'-Phosphate.

KCNQ2 mutations encompass a wide range of phenotypes, ranging from benign familial neonatal seizure to a clinical spectrum of early-onset epileptic encephalopathy that occurs in the early neonatal period. We report an infant with KCNQ2 encephalopathy presenting as neonatal seizure, initially controlled by two anticonvulsants. Electroencephalogram (EEG) showed repetitive multifocal epileptiform discharges, which remained similar after administration of intravenous pyridoxine injection. Seizure recurred at the age of 3 months preceded by an episode of minor viral infection, which occurred multiple times per day. No significant change in seizure frequency was observed after 5-day oral pyridoxine trial, but subsequently, there was dramatic seizure improvement with oral pyridoxal-5'-phosphate (PLP). We hope to alert clinicians that in patients with neonatal epileptic encephalopathy, particularly with known KCNQ2 mutations, intravenous injection of pyridoxine (preferably with EEG monitoring), followed by both oral trial of pyridoxine and PLP should be considered. KCNQ2 mutations should also be considered in vitamin B6-responsive patients.

Erratum: Erratum: KCNQ2 Encephalopathy and Responsiveness to Pyridoxal-5'-Phosphate.

[This corrects the article DOI: 10.1055/s-0040-1721384.].

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.

Genotype/phenotype correlation in 123 Chinese patients with Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is a multisystemic neurocutaneous disorder with autosomal dominant inheritance. We performed mutation analyses on 123 Chinese patients with "definite TSC" according to the latest diagnostic criteria. Pathogenic / likely-pathogenic variants were identified in 72.2% of all index patients (70/97), in which 35.7% (25/70) had TSC1 variants and 64.3% (45/70) had TSC2 variants. 84.5% (82/97) cases were sporadic and 15.5% (15/97) cases were familial. 62 unique variants were reported, in which 41.9% (26/62) were novel. Male patients had significantly more subependymal nodules (p=0.029) than females, whereas renal angiomyolipoma (p=0.032) occurred predominantly in females. Sporadic cases also had more renal angiomyolipoma (p=0.004), cortical tubers (p=0.008), hypopigmented macules (p=0.018) and fibrous cephalic plaques (p=0.028) than cases with known inheritance. Patients with TSC2 pathogenic variants were more likely to have mental retardation (p<0.001), cardiac rhabdomyoma (p=0.004), renal angiomyolipoma (p=0.006) and facial angiofibromas (p=0.026) than those with TSC1 pathogenic variants, while mutation-negative cases showed a mixed phenotype between those with TSC1 and TSC2 variants. There were no significant phenotypic differences between patients with and without TSC1/TSC2 variants, but TSC2 missense and in-frame variants were associated with higher frequencies of mental retardation (P<0.001), renal angiomyolipoma (p=0.001), cardiac rhabdomyoma (p=0.012) and facial angiofibroma (p=0.021) than those with TSC1 frameshift and splice site variants. Furthermore, a higher frequency of mental retardation (p=0.013) was observed in patients with TSC2 missense and in-frame variants than those with frameshift and splice site variants. All 14 antenatal-onset patients had cardiac rhabdomyoma. They had fewer seizures (p=0.028) than patients with paediatric-onset, but were more likely to have mental retardation (p=0.035) than individuals with adult-onset disease. Generally, paediatric-onset patients had more neurological manifestations, while initial presentations of adult-onset TSC were more diverse.

Successful Treatment of Drug-Resistant Seizures Secondary to Ring 20 Mosaicism with Perampanel as an Add-On Antiepileptic Drug.

We report a girl with drug-resistant seizures, progressive behavioral changes, and cognitive decline. Investigations showed abnormal EEG with frequent high-voltage bifrontotemporal sharp and slow waves, especially during sleep. Seizures were difficult to control, despite the usage of various antiepileptic drugs. Perampanel as an add-on antiepileptic drug appeared efficacious. Due to the recognizable pattern of seizures and EEG findings, a karyotype study was performed which revealed 46 chromosomes with a ring 20 chromosome mosaicism. Ring 20 chromosome is associated with drug-resistant refractory seizures, cognitive decline, and behavioral problems. This case highlights the difficulty and challenge faced in managing drug-resistant refractory seizures associated with ring 20 chromosome. While ring 20 chromosome is often underdiagnosed, one should have a high index of awareness and suspicion of such rare epilepsy syndrome, so that an early diagnosis can be made.

Investigation of Chromosomal Structural Abnormalities in Patients With Undiagnosed Neurodevelopmental Disorders.

Background: Structural variations (SVs) are various types of the genomic rearrangements encompassing at least 50 nucleotides. These include unbalanced gains or losses of DNA segments (copy number changes, CNVs), balanced rearrangements (such as inversion or translocations), and complex combinations of several distinct rearrangements. SVs are known to play a significant role in contributing to human genomic disorders by disrupting the protein-coding genes or the interaction(s) with cis-regulatory elements. Recently, different types of genome sequencing-based tests have been introduced in detecting various types of SVs other than CNVs and regions with absence of heterozygosity (AOH) with clinical significance. Method: In this study, we applied the mate-pair low pass (∼4X) genome sequencing with large DNA-insert (∼5 kb) in a cohort of 100 patients with neurodevelopmental disorders who did not receive informative results from a routine CNV investigation. Read-depth-based CNV analysis and chimeric-read-pairs analysis were used for CNV and SV analyses. The region of AOH was indicated by a simultaneous decrease in the rate of heterozygous SNVs and increase in the rate of homozygous SNVs. Results: First, we reexamined the 25 previously reported CNVs among 24 cases in this cohort. The boundaries of these twenty-five CNVs including 15 duplications and 10 deletions detected were consistent with the ones indicated by the chimeric-read-pairs analysis, while the location and orientation were determined in 80% of duplications (12/15). Particularly, one duplication was involved in complex rearrangements. In addition, among all the 100 cases, 10% of them were detected with rare or complex SVs (>10 Kb), and 3% were with multiple AOH (≥5 Mb) locating in imprinting chromosomes identified. In particular, one patient with an overall value of 214.5 Mb of AOH identified on 13 autosomal chromosomes suspected parental consanguinity. Conclusion: In this study, mate-pair low-pass GS resolved a significant proportion of CNVs with inconclusive significance, and detected additional SVs and regions of AOH in patients with undiagnostic neurodevelopmental disorders. This approach complements the first-tier CNV analysis for NDDs, not only by increasing the resolution of CNV detection but also by enhancing the characterization of SVs and the discovery of potential causative regions (or genes) contributory to could be complex in composition NDDs.

Detecting structural variations with precise breakpoints using low-depth WGS data from a single oxford nanopore MinION flowcell.

Structural variation (SV) is a major cause of genetic disorders. In this paper, we show that low-depth (specifically, 4×) whole-genome sequencing using a single Oxford Nanopore MinION flow cell suffices to support sensitive detection of SV, particularly pathogenic SV for supporting clinical diagnosis. When using 4× ONT WGS data, existing SV calling software often fails to detect pathogenic SV, especially in the form of long deletion, terminal deletion, duplication, and unbalanced translocation. Our new SV calling software SENSV can achieve high sensitivity for all types of SV and a breakpoint precision typically ± 100 bp; both features are important for clinical concerns. The improvement achieved by SENSV stems from several new algorithms. We evaluated SENSV and other software using both real and simulated data. The former was based on 24 patient samples, each diagnosed with a genetic disorder. SENSV found the pathogenic SV in 22 out of 24 cases (all heterozygous, size from hundreds of kbp to a few Mbp), reporting breakpoints within 100 bp of the true answers. On the other hand, no existing software can detect the pathogenic SV in more than 10 out of 24 cases, even when the breakpoint requirement is relaxed to ± 2000 bp.

ECNano: A cost-effective workflow for target enrichment sequencing and accurate variant calling on 4800 clinically significant genes using a single MinION flowcell.

BACKGROUND: The application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample. METHOD: We introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples. RESULTS: ECNano achieved deep on-target depth of coverage (DoC) at average > 100× and > 98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30× DoC. ECNano obtained an average read length of 1000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30× DoC. Clair-ensemble achieved > 99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days. CONCLUSION: We presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.

Old and new perspectives on Neurofibromatosis type 1: Clinical and molecular characterization of 832 patients from a single centre over 16 years.

Neurofibromatosis type 1 (NF1; OMIM #162200) is the commonest multi-systemic neurocutaneous tumour-predisposition disorder. It has an age-related complete penetrance but a highly variable inter- and intra-familial expressivity. This article summarizes the clinical features and molecular characteristics of 832 clinically or molecularly confirmed NF1 patients from 697 unrelated families recruited from a single centre in Hong Kong diagnosed during the 16 years period from Jan 2005 to Jan 2021. In this study, we have estimated the incidences of clinical features, reported on the molecular findings and explored new genotype-phenotype correlations.

HKG: an open genetic variant database of 205 Hong Kong cantonese exomes.

HKG is the first fully accessible variant database for Hong Kong Cantonese, constructed from 205 novel whole-exome sequencing data. There has long been a research gap in the understanding of the genetic architecture of southern Chinese subgroups, including Hong Kong Cantonese. HKG detected 196 325 high-quality variants with 5.93% being novel, and 25 472 variants were found to be unique in HKG compared to three Chinese populations sampled from 1000 Genomes (CHN). PCA illustrates the uniqueness of HKG in CHN, and the admixture study estimated the ancestral composition of HKG and CHN, with a gradient change from north to south, consistent with their geological distribution. ClinVar, CIViC and PharmGKB annotated 599 clinically significant variants and 360 putative loss-of-function variants, substantiating our understanding of population characteristics for future medical development. Among the novel variants, 96.57% were singleton and 6.85% were of high impact. With a good representation of Hong Kong Cantonese, we demonstrated better variant imputation using reference with the addition of HKG data, thus successfully filling the data gap in southern Chinese to facilitate the regional and global development of population genetics.

KBG syndrome in a Chinese population: A case series.

KBG syndrome (OMIM #148050) is an autosomal dominant neurodevelopmental disorder characterized by the presence of macrodontia of the permanent central upper incisors, characteristic facial features, delay in development, intellectual disability, short stature, and various skeletal abnormalities. Over 200 affected individuals have been described worldwide, though underdiagnosis is suspected because the characteristic features are variably present and affected individuals can have a mild phenotype. This case series provides a summary of the clinical and molecular characteristics of 10 Chinese KBG syndrome patients recruited from a single center. To our knowledge, this is the first case series for Chinese KBG patients. This case series aimed at exploring potential ethnicity-related variability in KBG syndrome.

Prenatal presentation in two fetuses with features of Beckwith Wiedemann syndrome-An unexpected diagnosis of androgenetic chimera and its clinical implications.

Beckwith Wiedemann Syndrome (BWS, OMIM 130650) is an imprinting disorder that may present antenatally with a constellation of sonographic features namely polyhydramnios, macrosomia, macroglossia, omphalocele, placental mesenchymal dysplasia, cardiomegaly, nephromegaly, fetal hydrops, and other rare anomalies. Paternal uniparental disomy in chromosome 11p15 imprinting region accounts for 20% of all BWS, and 8% among those were due to genome-wide paternal uniparental disomy (GWpUPD). GWpUPD is a rare condition and usually results in prenatal lethality. The 31 liveborns reported in the literature demonstrate female predominance in surviving GWpUPD. Here, we reported two prenatal cases which initially presented with features suggestive of BWS, which subsequently were confirmed to have GWpUPD. Further trio SNP genotyping analysis using SNP-based chromosomal microarray revealed androgenetic biparental chimera as the underlying cause. Finally, we highlighted the importance of recognizing GWpUPD as a possible cause in a fetus presenting with BWS phenotype, as it carried a different disease prognosis, tumor predisposition, manifestations of other imprinting disorders, and possibility in unmasking autosomal recessive disorders from the paternal alleles.

The first case report of Strømme syndrome in a Chinese patient: Expanding the phenotype and literature review.

Strømme syndrome (MIM #243605) is a rare autosomal recessive ciliopathy resulting from compound heterozygous or homozygous pathogenic alterations in the CENPF gene (# 600236). Although there are a number of case reports featuring individuals with clinically compatible Strømme syndrome, only 13 affected individuals had molecular confirmation worldwide. Herein, we report a 24 years old Chinese gentleman with molecularly confirmed Strømme syndrome with compound heterozygous pathogenic nonsense variants in NM_016343.3(CENPF):c.436C > T, p.(Gln146*) and c.9280C > T, p.(Arg3094*). He presented with microcephaly, unilateral microphthalmia, single central upper incisor and bilateral preaxial polydactyly. To our knowledge, this is the first reported Chinese individual with molecularly confirmed Strømme syndrome.