Ee Shien Tan

Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism.

Use of deferiprone for iron chelation in patients with transfusion‐dependent thalassaemia

Aim:  To conduct a retrospective case analysis of the clinical efficacy and adverse effects of deferiprone in our population.

Incidentalome from Genomic Sequencing: A Barrier to Personalized Medicine?

Background In Western cohorts, the prevalence of incidental findings (IFs) or incidentalome, referring to variants in genes that are unrelated to the patients primary condition, is between 0.86% and 8.8%. However, data on prevalence and type of IFs in Asian population is lacking. Methods In 2 cohorts of individuals with genomic sequencing performed in Singapore (total n = 377), we extracted and annotated variants in the 56 ACMG-recommended genes and filtered these variants based on the level of pathogenicity. We then analyzed the precise distribution of IFs, class of genes, related medical conditions, and potential clinical impact. Results We found a total of 41,607 variants in the 56 genes in our cohort of 377 individuals. After filtering for rare and coding variants, we identified 14 potential variants. After reviewing primary literature, only 4 out of the 14 variants were classified to be pathogenic, while an additional two variants were classified as likely pathogenic. Overall, the cumulative prevalence of IFs (pathogenic and likely pathogenic variants) in our cohort was 1.6%. Conclusion The cumulative prevalence of IFs through genomic sequencing is low and the incidentalome may not be a significant barrier to implementation of genomics for personalized medicine.

Tricho-hepato-enteric syndrome (THE-S): two cases and review of the literature

AbstractTricho-hepato-enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. We report two cases of East Asian descent with THE-S who had remained undiagnosed despite extensive investigations but were diagnosed on whole exome sequencing (WES). Both cases presented with chronic diarrhea, failure to thrive, and recurrent infections. Case 1 had posteriorly rotated low set ears, mild retrognathia, and fine curly hypopigmented hair. She was managed with prolonged total parenteral nutrition and intravenous immunoglobulin infusions. Case 2 had sparse coarse brown hair as well as multiple lentigines and café-au-lait macules. She was managed with amino acid-based formula. For both cases, routine investigations were inconclusive. WES in both cases showed biallelic truncating mutations in TTC37 (c.3507T>G;p.Y1169X and c.3601C>T;p.R1201X in case 1 and c.3507T>G;p.Y1169X and c.154G>T;p.E52X in case 2), suggesting a diagnosis of THE-S. Conclusion: We present novel mutations in the TTC37 gene in two individuals of East Asian descent with the rare THE-S, detected by WES. Future identification of patients with THE-S and establishing genotype-phenotype correlations will aid in counseling the patients and their families.What is Known:• Tricho-Hepato-Enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction.• Complex patients with diagnostic dilemmas undergo extensive investigations.What is New:• This is a report of novel mutations in TTC37 in individuals of East Asian descent.• Whole exome sequencing (WES) can be useful in certain complex cases with diagnostic dilemmas.

Cockayne Syndrome due to a maternally-inherited whole gene deletion of ERCC8 and a paternally-inherited ERCC8 exon 4 deletion.

Cockayne Syndrome (CS) is an autosomal recessive disorder that causes neurological regression, growth failure and dysmorphic features. We describe a Chinese female child with CS caused by deletions of exon 4 of ERCC8 on one chromosome and exons 1-12 on the other chromosome. By using chromosomal microarray, multiplex ligation-dependant probe analysis and long range PCR, we showed that she inherited a 277 kb deletion affecting the whole ERCC8 gene from the mother and a complex rearrangement resulting in deletion of exon 4 together with a 1,656 bp inversion of intron 4 from the father. A similar complex rearrangement has been reported in four unrelated Japanese CS patients. Analysis of the deletion involving exon 4 identified LINE and other repeat elements that may predispose the region to deletions, insertions and inversions. The patient also had insulin-dependent diabetes mellitus, a rare co-existing feature in patients with CS. More research will be needed to further understand the endocrine manifestations in CS patients.

Williams–Beuren syndrome in diverse populations

Williams–Beuren syndrome (WBS) is a common microdeletion syndrome characterized by a 1.5Mb deletion in 7q11.23. The phenotype of WBS has been well described in populations of European descent with not as much attention given to other ethnicities. In this study, individuals with WBS from diverse populations were assessed clinically and by facial analysis technology. Clinical data and images from 137 individuals with WBS were found in 19 countries with an average age of 11 years and female gender of 45%. The most common clinical phenotype elements were periorbital fullness and intellectual disability which were present in greater than 90% of our cohort. Additionally, 75% or greater of all individuals with WBS had malar flattening, long philtrum, wide mouth, and small jaw. Using facial analysis technology, we compared 286 Asian, African, Caucasian, and Latin American individuals with WBS with 286 gender and age matched controls and found that the accuracy to discriminate between WBS and controls was 0.90 when the entire cohort was evaluated concurrently. The test accuracy of the facial recognition technology increased significantly when the cohort was analyzed by specific ethnic population (P‐value < 0.001 for all comparisons), with accuracies for Caucasian, African, Asian, and Latin American groups of 0.92, 0.96, 0.92, and 0.93, respectively. In summary, we present consistent clinical findings from global populations with WBS and demonstrate how facial analysis technology can support clinicians in making accurate WBS diagnoses.

AB151. CHD7 variants identified by next-generation sequencing.

Background CHARGE syndrome is a genetic disorder with clinical features including ocular coloboma, heart defects, choanal atresia, retardation or developmental delay, genital hypoplasia, ear anomalies and deafness. Mutations in chromodomain helicase DNA binding protein 7 (CHD7) regulatory gene have been associated with this syndrome. CHD7 gene mutations accounted for more than half of patients with CHARGE syndrome. Advancement of next-generation sequencing technologies like the introduction of bench-top next-generation sequencers has enabled cost-effective and accurate detection of mutations in large genes or disorders which are difficult to diagnose clinically. Molecular diagnosis based solely on Sanger sequencing is time consuming and less efficient. The aim of this study was to test the ability of a targeted gene panel and bench-top sequencing to molecularly diagnose patients with CHARGE syndrome.

Urinary reducing substances in neonatal intrahepatic cholestasis caused by citrin deficiency

Neonatal cholestasis due to citrin deficiency is an autosomal recessive metabolic disorder caused by mutations in SLC25A13 gene. Mutations in this gene have a relatively high prevalence in East-Asian races compared to European or Afro-Caribbean races. Mutations in both sets of chromosomes often lead to self-limiting early onset cholestasis and growth retardation referred as neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). It is associated with a wide range of metabolic derangements including galactosemia and aminoacidemia, which can be detected on the newborn blood spot screening. Galactose, being a reducing sugar, can also be detected using Clinitest® (Clinitest® Reagent Tablets, Bayer Corporation, Diagnostics Division, Elkhart, IN, USA), a common screening test used in the work up of metabolic and hepatic diseases. In the western population classical galactosemia is often suspected when non glucose reducing substances are detected in the urine of infants with cholestasis. However in East-Asian races the prevalence of classical galactosemia is very low whilst galactosemia due to altered uridine diphosphate-galactose epimerase activity in NICCD is more common. We present a case of NICCD in an East-Asian infant with cholestasis and persistently positive urine reducing substance. Conclusion: NICCD deficiency should be considered as a differential diagnosis in any infant with cholestasis and persistently positive urinary reducing substances.

The Clinical Effects of Fermented Papaya Preparation® (FPP®) on Oxidative Stress in Patients with HbE/ β -Thalassaemia

Background: Red blood cells (RBC) of patients with thalassaemia are under continuous oxidative stress. Fermented papaya preparation® (FPP®) has been shown to have an antioxidative effect and is postulated to reduce the oxidative stress on RBC. Objective: To study the clinical effects of FPP® treatment in patients with HbE/β-thalassaemia on RBC indices, oxidative stress and quality of life scores. Method: Patients with HbE/β-thalassaemia who do not receive regular blood transfusion were included in the study and were given FPP® daily (3gm 2 times a day) for 12 weeks. Peripheral blood samples were obtained at the initiation of the study and at 4-weekly intervals thereafter for a period of 12 weeks. The following parameters were measured: Haemoglobin (Hb), mean corpuscular volume (MCV), reticulocyte count; Oxidation studies: production of reactive oxygen species (ROS) and intracellular glutathione content (GSH), spontaneously and in response to oxidative stress; Quality of life (QoL) at the start and at the end of 12 weeks using health survey questionnaires. Results: Seven patients (5 females and 2 males) were recruited to the study from January 2006 to April 2006. Median age of the study population was 19 years (range 4 to 27yrs). In vitro analyses showed production of significantly less ROS and more GSH following treatment. There was no significant difference in the Hb, MCV, reticulocyte count, clinical parameters or QoL scores. FPP® was well tolerated by all the patients. Conclusion: Although oxidative stress parameters were decreased, FPP® did not have any significant effect on the Hb levels or QoL. Longer studies on larger sample size are required to study the long-term clinical effect of FPP® on clinical parameters in patients with Hb E/β-thalassaemia.