Cluster analysis and visualisation of electronic health records data to identify undiagnosed patients with rare genetic diseases.

Rare genetic diseases affect 5-8% of the population but are often undiagnosed or misdiagnosed. Electronic health records (EHR) contain large amounts of data, which provide opportunities for analysing and mining. Data mining, in the form of cluster analysis and visualisation, was performed on a database containing deidentified health records of 1.28 million patients across 3 major hospitals in Singapore, in a bid to improve the diagnostic process for patients who are living with an undiagnosed rare disease, specifically focusing on Fabry Disease and Familial Hypercholesterolaemia (FH). On a baseline of 4 patients, we identified 2 additional patients with potential diagnosis of Fabry disease, suggesting a potential 50% increase in diagnosis. Similarly, we identified > 12,000 individuals who fulfil the clinical and laboratory criteria for FH but had not been diagnosed previously. This proof-of-concept study showed that it is possible to perform mining on EHR data albeit with some challenges and limitations.

A novel intronic variant in ROBO3 associated with horizontal gaze palsy with progressive scoliosis: case report and literature review.

Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare, autosomal recessive inherited disorder caused by mutations in ROBO3 gene. The clinical features of HGPPS include horizontal gaze palsy, progressive scoliosis, other oculomotor abnormalities such as strabismus and nystagmus. Whole-exome sequencing (WES) is used to diagnose rare Mendelian disorders, when routine standard tests have failed to make a formal pathological diagnosis. However, WES may identify variants of uncertain significance (VUS) that may add further ambiguity to the diagnosis. We report the case of a 4-year-old boy with horizontal gaze palsy, progressive scoliosis, microcephaly, and mild developmental delay. WES identified an intronic VUS in ROBO3 gene. We performed minigene splicing functional analysis to confirm the pathogenicity of this VUS. This report illustrates that WES data analysis with supportive functional analysis provides an effective approach to improve the diagnostic yield for unsolved clinical cases. This case also highlights the phenotypic heterogeneity in patients with HGPPS.

RETINITIS PIGMENTOSA ASSOCIATED WITH THE EYS C2139Y VARIANT : An Important Cause of Blindness in East Asian Populations.

PURPOSE: The study aimed to describe the phenotypic features of retinitis pigmentosa (RP) associated with the previously described EYS C2139Y variant in Singaporeans and establish the importance of this variant as a prevalent cause of RP among East Asians. METHODS: A clinical phenotyping and exome-sequencing study was conducted on consecutive patients with nonsyndromic RP. Epidemiological analysis was performed using Singaporean and global population-based genetic data. RESULTS: A study of 150 consecutive unrelated individuals with nonsyndromic RP found that 87 (58%) of cases had plausible genotypes. A previously described missense variant in the EYS gene, 6416G>A (C2139Y), occurred heterozygously or homozygously in 17 of 150 families (11.3%), all with autosomal recessive RP. Symptom onset in EYS C2139Y-related RP ranged from 6 to 45 years, with visual acuity ranging from 20/20 at 21 years to no light perception by 48 years. C2139Y-related RP had typical findings, including sectoral RP in cases with EYS E2703X in trans . The median age at presentation was 45 years and visual fields declined to less than 20° (Goldmann V4e isopter) by age 65 years. Intereye correlation for visual acuity, fields, and ellipsoid band width was high (r 2 = 0.77-0.95). Carrier prevalence was 0.66% (allele frequency of 0.33%) in Singaporean Chinese and 0.34% in East Asians, suggesting a global disease burden exceeding 10,000 individuals. CONCLUSION: The EYS C2139Y variant is common in Singaporean RP patients and other ethnic Chinese populations. Targeted molecular therapy for this single variant could potentially treat a significant proportion of RP cases worldwide.

DEGS1 -related leukodystrophy: a clinical report and review of literature.

BACKGROUND: Leukodystrophies are a heterogeneous group of disorders affecting the white matter of the central nervous system, with or without affecting the peripheral nervous system. Biallelic variants in DEGS1 , coding for desaturase 1 (Des1) protein, were recently reported to be associated with hypomyelinating leukodystrophy (HLD), a subclass of leukodystrophies where the formation of the myelin sheath is affected. METHODS: Genomic sequencing was performed on our index patient with severe developmental delay, severe failure to thrive, dystonia, seizures, and hypomyelination on brain imaging. Sphingolipid analysis was performed and dihydroceramide/ceramide (dhCer/Cer) ratios were obtained by the measurement of ceramide and dihydroceramide species. RESULTS: A homozygous missense variant was identified in DEGS1 (c.565A > G:p Asn189Asp). The identified DEGS1 variant has been annotated as "conflicting reports of pathogenicity" on ClinVar. Follow-up sphingolipid analysis on our patient showed significantly raised dhCer/Cer and this was consistent with dysfunction of the Des1 protein, providing additional evidence to support the pathogenicity of this variant. CONCLUSION: While rare, pathogenic variants in DEGS1 should be considered in patients with HLD phenotype. To date, 25 patients have been reported across four studies on DEGS1 -related HLD, and, in this report, we summarize the literature. More such reports will enable deeper phenotypic characterization of this disorder.

Diverse Clinical Manifestations of Cardiofaciocutaneous Syndrome Type 3 in Two Patients from South East Asia.

Background: Cardiofaciocutaneous syndrome (CFCS) is a rare genetic condition caused by mutations in BRAF, KRAS, MAP2K1, or MAP2K2. It is characterized by ectodermal abnormalities, cardiac defects, intellectual disability, and distinct craniofacial features. CFCS falls under a group of conditions caused by mutations in the RAS/MAPK pathway called RASopathies which share many features. In particular, CFCS has significant phenotypic overlaps with Costello syndrome (CS) and Noonan syndrome (NS). Objective: The aim of this study was to assess the patients‧ phenotypic features for syndromic disorders and evaluate the use of molecular testing to clarify the clinical diagnosis. Method: The patients were recruited for genetic testing with written informed consent. Genomic DNA from venous blood was sequenced and potential variants were identified via targeted next-generation sequencing. Their phenotypic features were compared with other CFCS cases carrying pathogenic variants in the same gene. Results and Discussion: One patient had a de novo variant (c.370C>T; p.P124S) in MAP2K1 and presented with mild and typical features which do not significantly affect her quality of life. The second patient presented with severe features, including failure to thrive, feeding difficulties, epileptic spasms, septal hypertrophy, and global developmental delay, and developed chronic lung disease and sequelae from multiple infections. She had a severe disease course and severe global developmental delay. The discovery of a de novo variant (c.371C>A; p.P124Q) in MAP2K1, which had been reported in another patient with a similar phenotype, clarifies her clinical diagnosis. Her presentations add to existing reports that support expanding the CFCS phenotype to include features previously thought to be more suggestive of CS. Conclusion: The genetic findings for the 2 patients affirm the use of identified gene mutations to confirm the clinical diagnosis of syndromic disorders and add to the phenotypic spectrum of CFCS.

Reduced resource utilization with early use of next-generation sequencing in rare genetic diseases in an Asian cohort.

Children with genetic diseases endure a prolonged and costly "diagnostic odyssey." The use of whole exome sequencing (WES) and whole genome sequencing (WGS) has improved the diagnosis rate, ending the odyssey. However, the additional costs associated WES/WGS has impeded their adoption in Asian settings. We aim to estimate the expected change to the mean number of diagnostic tests used, and the associated costs from a decision to use WES early in the diagnostic pathways of pediatric phenotypes, as compared to Existing Practice. Retrospective data from a patient cohort recruited under the Singapore Undiagnosed Disease Program from a tertiary hospital in Singapore, for the period October 2004 to September 2020, was analyzed. Four phenotype-specific subgroups were used: multiple congenital anomalies (MCA) without developmental delay; global developmental delay (GDD); neuromuscular disorder (NMD) and primary immunodeficiency disorder (PID). Patients had undergone a traditional diagnostic pathway and received a diagnosis either through clinical exome or WES or WGS. A costs only analysis was performed, by tabulating the outcomes "test quantity" and "test costs" incurred by patients. The outcomes were compared with alternate diagnostic pathways which incorporates the early introduction of WES trio testing. To include uncertainty in cost outcomes, simulation studies were done on uncertain parameters. Cost outcomes are reported in Singapore dollars (S$). The 92 included patients had MCA (n = 48), GDD (n = 29), NMD (n = 10), or PID (n = 5). Patients were aged between 18 days and 26 years, 52.2% were males. The majority were of Chinese ethnicity (81.5%). If patients had access to WES directly, test quantity reduced by 97.38% for MCA, 96.98% for GDD, 96.56% for NMD, and 99.84% for PID. The expected cost savings per patient were $5940 for MCA (US$4433), $5342 for GDD (US$3986), $4622 for NMD (US$3449), and $58,497 for PID (US$43,654). Uncertainty assessment for MCA and GDD patients showed a respective likelihood of 86.9% and 97.4% for cost savings. Adoption of alternate diagnostic pathways with early WES in selected pediatric subgroups are likelt to reduce costs, when compared to Existing Practice. Benefits arising from earlier diagnosis, and the potential cost savings could mitigate the large initial cost of implementing WES in Asian settings.

Family history assessment significantly enhances delivery of precision medicine in the genomics era.

BACKGROUND: Family history has traditionally been an essential part of clinical care to assess health risks. However, declining sequencing costs have precipitated a shift towards genomics-first approaches in population screening programs rendering the value of family history unknown. We evaluated the utility of incorporating family history information for genomic sequencing selection. METHODS: To ascertain the relationship between family histories on such population-level initiatives, we analysed whole genome sequences of 1750 research participants with no known pre-existing conditions, of which half received comprehensive family history assessment of up to four generations, focusing on 95 cancer genes. RESULTS: Amongst the 1750 participants, 866 (49.5%) had high-quality standardised family history available. Within this group, 73 (8.4%) participants had an increased family history risk of cancer (increased FH risk cohort) and 1 in 7 participants (n = 10/73) carried a clinically actionable variant inferring a sixfold increase compared with 1 in 47 participants (n = 17/793) assessed at average family history cancer risk (average FH risk cohort) (p = 0.00001) and a sevenfold increase compared to 1 in 52 participants (n = 17/884) where family history was not available (FH not available cohort) (p = 0.00001). The enrichment was further pronounced (up to 18-fold) when assessing only the 25 cancer genes in the American College of Medical Genetics (ACMG) Secondary Findings (SF) genes. Furthermore, 63 (7.3%) participants had an increased family history cancer risk in the absence of an apparent clinically actionable variant. CONCLUSIONS: These findings demonstrate that the collection and analysis of comprehensive family history and genomic data are complementary and in combination can prioritise individuals for genomic analysis. Thus, family history remains a critical component of health risk assessment, providing important actionable data when implementing genomics screening programs. TRIAL REGISTRATION: ClinicalTrials.gov NCT02791152 . Retrospectively registered on May 31, 2016.

Genetic landscape of congenital disorders in patients from Southeast Asia: results from sequencing using a gene panel for Mendelian phenotypes.

OBJECTIVE: To test the utility and diagnostic yield of a medical-exome gene panel for identifying pathogenic variants in Mendelian disorders. METHODS: Next-generation sequencing was performed with the TruSight One gene panel (targeting 4813 genes) followed by MiSeq sequencing on 216 patients who presented with suspected genetic disorders as assessed by their attending physicians. RESULTS: There were 56 pathogenic and 36 likely pathogenic variants across 57 genes identified in 87 patients. Causal mutations were more likely to be truncating and from patients with a prior clinical diagnosis. Another 18 promising variants need further evaluation for more evidence to meet the requirement for potential upgrade to pathogenic. Forty-five of the 92 clinically significant variants were novel. CONCLUSION: The 40.3% positive yield compares favourably with similar studies using either this panel or whole exome sequencing, demonstrating that large gene panels could be a good alternative to whole exome sequencing for quick genetic confirmation of Mendelian disorders.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

Implementation of genomics in medical practice to deliver precision medicine for an Asian population.

Whilst the underlying principles of precision medicine are comparable across the globe, genomic references, health practices, costs and discrimination policies differ in Asian settings compared to the reported initiatives involving European-derived populations. We have addressed these variables by developing an evolving reference base of genomic and phenotypic data and a framework to return medically significant variants to consenting research participants applicable for the Asian context. Targeting 10,000 participants, over 2000 Singaporeans, with no known pre-existing health conditions, have consented to an extensive clinical health screen, family health history collection, genome sequencing and ongoing follow-up. Genomic variants in a subset of genes associated with Mendelian disorders and drug responses are analysed using an in-house bioinformatics pipeline. A multidisciplinary team reviews the classification of variants and a research report is generated. Medically significant variants are returned to consenting participants through a bespoke return-of-result genomics clinic. Variant validation and subsequent clinical referral are advised as appropriate. The design and implementation of this flexible learning framework enables a cohort of detailed phenotyping and genotyping of healthy Singaporeans to be established and the frequency of disease-causing variants in this population to be determined. Our findings will contribute to international precision medicine initiatives, bridging gaps with ethnic-specific data and insights from this understudied population.

Experience of Asian males communicating cardiac genetic risk within the family.

The genetic nature of an inherited cardiac condition (ICC) places first- and second-degree relatives at risk of cardiac complications and sudden death, even in the absence of symptoms. Communication of cardiac genetic risk information allows at-risk relatives to clarify, manage, and potentially prevent ICC-associated risks through cardiac screening. Literature regarding family communication of genetic risk information are predominantly based on Western populations, with limited insight into the Asian experience. This qualitative exploratory study provides a male perspective into the communication of ICC risks within families in Singapore. Eight male participants with clinically diagnosed cardiomyopathy, who had all received genetic counseling, were recruited. A phenomenological perspective was used to identify emergent themes from semi-structured interviews. In this study, most participants recalled their healthcare professional's emphasis on family communication. Notably, participants revealed that at-risk relatives were not accessing screening, and many described family members as currently asymptomatic and "healthy." These findings coincide with documented Asian beliefs regarding perceptions of health, which have important implications for the provision of genetic counseling support within Asian communities, especially in facilitating family communication such that at-risk relatives are informed about their ICC risks and available management options.