Gordon Ka Chun Leung

Noonan syndrome in diverse populations

Noonan syndrome (NS) is a common genetic syndrome associated with gain of function variants in genes in the Ras/MAPK pathway. The phenotype of NS has been well characterized in populations of European descent with less attention given to other groups. In this study, individuals from diverse populations with NS were evaluated clinically and by facial analysis technology. Clinical data and images from 125 individuals with NS were obtained from 20 countries with an average age of 8 years and female composition of 46%. Individuals were grouped into categories of African descent (African), Asian, Latin American, and additional/other. Across these different population groups, NS was phenotypically similar with only 2 of 21 clinical elements showing a statistically significant difference. The most common clinical characteristics found in all population groups included widely spaced eyes and low‐set ears in 80% or greater of participants, short stature in more than 70%, and pulmonary stenosis in roughly half of study individuals. Using facial analysis technology, we compared 161 Caucasian, African, Asian, and Latin American individuals with NS with 161 gender and age matched controls and found that sensitivity was equal to or greater than 94% for all groups, and specificity was equal to or greater than 90%. In summary, we present consistent clinical findings from global populations with NS and additionally demonstrate how facial analysis technology can support clinicians in making accurate NS diagnoses. This work will assist in earlier detection and in increasing recognition of NS throughout the world.

Mechanisms for the Generation of Two Quadruplications Associated with Split-Hand Malformation

Germline copy‐number variants (CNVs) involving quadruplications are rare and the mechanisms generating them are largely unknown. Previously, we reported a 20‐week gestation fetus with split‐hand malformation; clinical microarray detected two maternally inherited triplications separated by a copy‐number neutral region at 17p13.3, involving BHLHA9 and part of YWHAE. Here, we describe an 18‐month‐old male sibling of the previously described fetus with split‐hand malformation. Custom high‐density microarray and digital droplet PCR revealed the copy‐number gains were actually quadruplications in the mother, the fetus, and her later born son. This quadruplication‐normal‐quadruplication pattern was shown to be expanded from the triplication‐normal‐triplication CNV at the same loci in the maternal grandmother. We mapped two breakpoint junctions and demonstrated that both are mediated by Alu repetitive elements and identical in these four individuals. We propose a three‐step process combining Alu‐mediated replicative‐repair‐based mechanism(s) and intergenerational, intrachromosomal nonallelic homologous recombination to generate the quadruplications in this family.

De novo large rare copy-number variations contribute to conotruncal heart disease in Chinese patients

Conotruncal heart anomalies (CTDs) are particularly prevalent congenital heart diseases (CHD) in Hong Kong. We surveyed large (>500 kb), rare (<1% frequency in controls) copy-number variations (CNVs) in Chinese patients with CTDs to identify potentially disease-causing variations. Adults who tested negative for 22q11.2 deletions were recruited from the adult CHD clinic in Hong Kong. Using a stringent calling criteria, high-confidence CNV calls were obtained, and a large control set comprising 3,987 Caucasian and 1,945 Singapore Chinese subjects was used to identify rare CNVs. Ten large rare CNVs were identified, and 3 in 108 individuals were confirmed to harbour de novo CNVs. All three patients were syndromic with a more complex phenotype, and each of these CNVs overlapped regions likely to be important in CHD. One was a 611 kb deletion at 17p13.3, telomeric to the Miller–Dieker syndrome (MDS) critical region, overlapping the NXN gene. Another was a 5 Mb deletion at 13q33.3, within a previously described critical region for CHD. A third CNV, previously unreported, was a large duplication at 2q22.3 overlapping the ZEB2 gene. The commonly reported 1q21.1 recurrent duplication was not observed in this Chinese cohort. We provide detailed phenotypic and genotypic descriptions of large rare genic CNVs that may represent CHD loci in the East Asian population. Larger samples of Chinese origin will be required to determine whether the genome-wide distribution differs from that found in predominantly European CHD cohorts.

Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies

RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.

CFTR founder mutation causes protein trafficking defects in Chinese patients with cystic fibrosis

Cystic fibrosis (CF) is a rare condition in Asians. Since 1985, only about 30 Chinese patients have been reported with molecular confirmation.

Paternal uniparental disomy of chromosome 19 in a pair of monochorionic diamniotic twins with dysmorphic features and developmental delay

Background We report here clinical, cytogenetic and molecular data for a pair of monochorionic diamniotic twins with paternal isodisomy for chromosome 19. Both twins presented with dysmorphic features and global developmental delay. This represents, to our knowledge, the first individual human case of paternal uniparental disomy for chromosome 19 (UPD19). Methods Whole-exome sequencing, together with conventional karyotype and SNP array analysis were performed along with genome-wide DNA methylation array for delineation of the underlying molecular defects. Results Conventional karyotyping on amniocytes and lymphocytes showed normal karyotypes for both twins. Whole-exome sequencing did not identify any pathogenic sequence variants but >5000 homozygous exonic variants on chromosome 19, suggestive of UPD19. SNP arrays on blood and buccal DNA both showed paternal isodisomy for chromosome 19. Losses of imprinting for known imprinted genes on chromosome 19 were identified, including ZNF331, PEG3, ZIM2 and MIMT1. In addition, imprinting defects were also identified in genes located on other chromosomes, including GPR1-AS, JAKMP1 and NHP2L1. Conclusion Imprinting defects are the most likely cause for the dysmorphism and developmental delay in this first report of monozygotic twins with UPD19. However, epigenotype-phenotype correlation will require identification of additional individuals with UPD19 and further molecular analysis.

Okur-Chung neurodevelopmental syndrome: Eight additional cases with implications on phenotype and genotype expansion

Okur‐Chung syndrome is a neurodevelopmental condition attributed to germline CSNK2A1 pathogenic missense variants. We present 8 unreported subjects with the above syndrome, who have recognizable dysmorphism, varying degrees of developmental delay and multisystem involvement. Together with 6 previously reported cases, we present a case series of 7 female and 7 male subjects, highlighting the recognizable facial features of the syndrome (microcephaly, hypertelorism, epicanthic fold, ptosis, arched eyebrows, low set ears, ear fold abnormality, broad nasal bridge and round face) as well as frequently occurring clinical features including neurodevelopmental delay (93%), gastrointestinal (57%), musculoskeletal (57%) and immunological (43%) abnormalities. The variants reported in this study are evolutionary conserved and absent in the normal population. We observed that the CSNK2A1 gene is relatively intolerant to missense genetic changes, and most variants are within the protein kinase domain. All except 1 variant reported in this cohort are spatially located on the binding pocket of the holoenzyme. We further provide key recommendations on the management of Okur‐Chung syndrome. To conclude, this is the second case series on Okur‐Chung syndrome, and an in‐depth review of the phenotypic features and genomic findings of the condition with suggestions on clinical management.

Identifying the genetic causes for prenatally diagnosed structural congenital anomalies (SCAs) by whole-exome sequencing (WES)

BackgroundWhole-exome sequencing (WES) has become an invaluable tool for genetic diagnosis in paediatrics. However, it has not been widely adopted in the prenatal setting. This study evaluated the use of WES in prenatal genetic diagnosis in fetuses with structural congenital anomalies (SCAs) detected on prenatal ultrasound.MethodThirty-three families with fetal SCAs on prenatal ultrasonography and normal chromosomal microarray results were recruited. Genomic DNA was extracted from various fetal samples including amniotic fluid, chorionic villi, and placental tissue. Parental DNA was extracted from peripheral blood when available. We used WES to sequence the coding regions of parental-fetal trios and to identify the causal variants based on the ultrasonographic features of the fetus.ResultsPathogenic mutations were identified in three families (n = 3/33, 9.1%), including mutations in DNAH11, RAF1 and CHD7, which were associated with primary ciliary dyskinesia, Noonan syndrome, and CHARGE syndrome, respectively. In addition, variants of unknown significance (VUSs) were detected in six families (18.2%), in which genetic changes only partly explained prenatal features.ConclusionWES identified pathogenic mutations in 9.1% of fetuses with SCAs and normal chromosomal microarray results. Databases for fetal genotype-phenotype correlations and standardized guidelines for variant interpretation in prenatal diagnosis need to be established to facilitate the use of WES for routine testing in prenatal diagnosis.

Mosaic KRAS mutation in a patient with encephalocraniocutaneous lipomatosis and renovascular hypertension

Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON, Canada Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada Department of Radiology, Queen Mary Hospital, Hong Kong Department of Health, Clinical Genetic Service, Hong Kong Division of Paediatric Surgery, Department of Surgery, The University of Hong Kong, Hong Kong Correspondence Brian Hon-Yin Chung, Department of Paediatrics and Adolescent Medicine, 102 Pokfulam Road, Hong Kong. Email: bhychung@hku.hk and Kym M. Boycott, Department of Genetics, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON, Canada K1H 8L1. Email: kboycott@cheo.on.ca Funding information Baxter and Alma Ricard Foundation, Grant/Award Number: Doctoral Research Award; Canadian Institutes of Health Research; Care4Rare Canada Consortium; Children’s Hospital of Eastern Ontario Foundation; Genome Canada; Génome Québec; Ontario Genomics Institute; Ontario Research Fund; The Hong Kong Society for the Relief of Disabled Children

A novel patient with an attenuated Costello syndrome phenotype due to an HRAS mutation affecting codon 146-Literature review and update

De novo germline mutations in HRAS cause Costello syndrome, with >95% of the mutations causing Costello syndrome affecting amino acid position 12 (p.Gly12) or 13 (p.Gly13). We report on a patient with de novo missense mutation causing an amino acid change at codon 146 of HRAS, c.436G > C:p.Ala146Pro, who presented with subtle dysmorphic features, failure to thrive, global developmental delay, and hypertrophic obstructive cardiomyopathy. Mutations affecting codon 146 are observed in <1% of patients with Costello syndrome. From literature search, there were only two other patients reported with mutations involving the same location. We summarized and updated their findings, and discussed evidence to show that these patients with less obvious signs of Costello syndrome may not necessarily run a more benign clinical course.