Alan Ma

Extreme growth failure is a common presentation of ligase IV deficiency

Ligase IV syndrome is a rare differential diagnosis for Nijmegen breakage syndrome owing to a shared predisposition to lympho‐reticular malignancies, significant microcephaly, and radiation hypersensitivity. Only 16 cases with mutations in LIG4 have been described to date with phenotypes varying from malignancy in developmentally normal individuals, to severe combined immunodeficiency and early mortality. Here, we report the identification of biallelic truncating LIG4 mutations in 11 patients with microcephalic primordial dwarfism presenting with restricted prenatal growth and extreme postnatal global growth failure (average OFC −10.1 s.d., height −5.1 s.d.). Subsequently, most patients developed thrombocytopenia and leucopenia later in childhood and many were found to have previously unrecognized immunodeficiency following molecular diagnosis. None have yet developed malignancy, though all patients tested had cellular radiosensitivity. A genotype–phenotype correlation was also noted with position of truncating mutations corresponding to disease severity. This work extends the phenotypic spectrum associated with LIG4 mutations, establishing that extreme growth retardation with microcephaly is a common presentation of bilallelic truncating mutations. Such growth failure is therefore sufficient to consider a diagnosis of LIG4 deficiency and early recognition of such cases is important as bone marrow failure, immunodeficiency, and sometimes malignancy are long term sequelae of this disorder.

CHARGE syndrome: a review.

CHARGE syndrome is a complex genetic syndrome, owing to the wide range of tissues/systems affected by mutations in the CHD7 gene. In this review, we discuss the diagnosis, clinical features and management of CHARGE syndrome.

Sporadic and Familial Congenital Cataracts: Mutational Spectrum and New Diagnoses Using Next‐Generation Sequencing

Congenital cataracts are a significant cause of lifelong visual loss. They may be isolated or associated with microcornea, microphthalmia, anterior segment dysgenesis (ASD) and glaucoma, and there can be syndromic associations. Genetic diagnosis is challenging due to marked genetic heterogeneity. In this study, next‐generation sequencing (NGS) of 32 cataract‐associated genes was undertaken in 46 apparently nonsyndromic congenital cataract probands, around half sporadic and half familial cases. We identified pathogenic variants in 70% of cases, and over 68% of these were novel. In almost two‐thirds (20/33) of these cases, this resulted in new information about the diagnosis and/or inheritance pattern. This included identification of: new syndromic diagnoses due to NHS or BCOR mutations; complex ocular phenotypes due to PAX6 mutations; de novo autosomal‐dominant or X‐linked mutations in sporadic cases; and mutations in two separate cataract genes in one family. Variants were found in the crystallin and gap junction genes, including the first report of severe microphthalmia and sclerocornea associated with a novel GJA8 mutation. Mutations were also found in rarely reported genes including MAF, VIM, MIP, and BFSP1. Targeted NGS in presumed nonsyndromic congenital cataract patients provided significant diagnostic information in both familial and sporadic cases.

Further delineation of the KAT6B molecular and phenotypic spectrum

KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.

Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.

The Immune Phenotype of Patients with CHARGE Syndrome.

BACKGROUND Recurrent sinopulmonary infections are common in children with CHARGE (Coloboma, Heart disease, choanal Atresia, growth/mental Retardation, Genitourinary malformations, Ear abnormalities) syndrome, but no prospective studies on immune function have been conducted. OBJECTIVE This study aims to examine and compare the immune phenotype of patients with CHARGE syndrome to those with 22q11.2 deletion and healthy controls. METHODS A total of 21 patients attended a multidisciplinary CHARGE clinic. All patients had CHD7 mutational analysis performed. Patients with CHARGE syndrome had lymphocyte subsets, immunoglobulins (IgG, A, M), functional protein, and polysaccharide vaccine responses measured at initial evaluation. A total of 55 healthy controls were prospectively recruited, whereas 40 patients with 22q11.2 deletion were retrospectively identified through medical records. A separate analysis compared serial lymphocyte counts and ionized calcium levels between patients with CHARGE syndrome and those with 22q11.2 deletion in the first 72 months of life. RESULTS Despite recurrent childhood ear and chest infections, only 2 children with CHARGE syndrome had an identifiable immune defect (reduced serum IgA). In contrast, T-cell lymphopenia, low immunoglobulin levels, and specific antibody deficiency were noted in patients with 22q11.2 deletion. A greater proportion of patients with 22q11.2 deletion had persistent lymphopenia (57% vs 30%) and hypocalcemia (60% vs 37.5%) compared with patients with CHARGE syndrome in the first 72 months of life. CONCLUSIONS Although phenotypic overlap exists between CHARGE and 22q11.2 deletion syndromes, no significant immune defects were detected in this cohort of patients with CHARGE syndrome at the time of testing. Lymphopenia and hypocalcemia occur in both conditions early in life, but is more pronounced in patients with 22q11.2 deletion.

New mutations in GJA8 expand the phenotype to include total sclerocornea

This project expands the disease spectrum for mutations in GJA8 to include total sclerocornea, rudimentary lenses and microphthalmia, in addition to this genes previously known role in isolated congenital cataracts. Ophthalmic findings revealed bilateral total sclerocornea in 3 probands, with small abnormal lenses in 2 of the cases, and cataracts and microphthalmia in 1 case. Next‐generation sequencing revealed de novo heterozygous mutations affecting the same codon of GJA8 : (c.281G>A; p.(Gly94Glu) and c.280G>C; p.(Gly94Arg)) in 2 of the probands, in addition to the c.151G>A; p.(Asp51Asn) mutation we had previously identified in the third case. In silico analysis predicted all of the mutations to be pathogenic. These cases show that deleterious, heterozygous mutations in GJA8 can lead to a severe ocular phenotype of total sclerocornea, abnormal lenses, and/or cataracts with or without microphthalmia, broadening the phenotype associated with this gene. GJA8 should be included when investigating patients with the severe anterior segment abnormality of total sclerocornea.

Targeted knockout of a chemokine-like gene increases anxiety and fear responses

Significance Emotion-related responses, such as fear and anxiety, are important behavioral phenomena in most animal species, as well as in humans. However, the underlying mechanisms of fear and anxiety in animals and in humans are still largely unknown, and anxiety disorders continue to represent a large unmet medical need in the human clinic. Animal models may speed up discovery of these mechanisms and may also lead to betterment of human health. Herein, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of sam2. We observed increased anxiety-related responses in both zebrafish and mouse knockout models. Taken together, these results support a crucial and evolutionarily conserved role of sam2 in regulating anxiety-like behavior. Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2. We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

BackgroundBeckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR.ResultsSingle nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p < 0.017); however, the relationship was not significant when the Bonferroni correction for multiple testing was applied (significance, p = 0.0036). None of the remaining 13 SNVs were significantly different in the two populations tested. The DNMT1 locus was screened in 53 BWS cases, and three rare missense variants were identified in each of three patients: rs138841970: C>T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity.ConclusionsThis study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibly via a maternal effect in the early embryo. Larger cohort studies are warranted to further interrogate the relationship between impaired MTHFR enzymatic activity attributable to MTHFR rs1801133: C>T, dietary folate intake and BWS.

Prenatal sonographic features of cranioectodermal dysplasia

Department of Obstetrics and Gynaecology at Nepean Hospital, New South Wales, Australia Department of Clinical Genetics, Nepean Hospital, New South Wales, Australia Disciplines of Genetic Medicine, Child and Adolescent Health, Children’s Hospital Westmead Clinical School, Sydney University, New South Wales, Australia Medical Sonography, School of Health, Medical and Applied Science, Central Queensland University, Australia Medical School, College of Medicine, Biology and Environment, The Australian National University, Australia *Correspondence to: T. Muttusamy, E-mail: thanursini_246@hotmail.com