Lesley Turner

Phenotypic Spectrum Associated with De Novo and Inherited Deletions and Duplications at 16p11.2 in Individuals Ascertained for Diagnosis of Autism Spectrum Disorder.

Background Recurrent microdeletions and microduplications of ∼555 kb at 16p11.2 confer susceptibility to autism spectrum disorder (ASD) in up to 1% of ASD patients. No physical or behavioural features have been identified that distinguish these individuals as having a distinct ASD subtype, but clinical data are limited. Methods We report five autistic probands identified by microarray analysis with copy number variation (CNV) of 16p11.2 (three deletions, two duplications). Each patient was assessed for ASD and dysmorphic features. We also describe a deletion positive 26-month-old female who has developmental delay (DD) and autistic features. Results Proband 1 (female with ASD, de novo deletion) is not dysmorphic. Proband 2 (male with autism, de novo deletion) and proband 3 and his brother (males with autism, inherited deletions) are dysmorphic, but the two probands do not resemble one another. The mother of proband 3 has mild mental retardation (MR), minor dysmorphism and meets the criteria for ASD. Proband 4 (dysmorphic autistic male, de novo duplication) had a congenital diaphragmatic hernia. Proband 5 (non-dysmorphic ASD female with a duplication) has two apparently healthy duplication positive relatives. Probands 1 and 2 have deletion negative siblings with ASD and Asperger syndrome, respectively. Proband 6 (a female with DD and an inherited duplication) is dysmorphic, but has oligohydramnios sequence. Conclusions The phenotypic spectrum associated with CNV at 16p11.2 includes ASD, MR/DD and/or possibly other primary psychiatric disorders. Compared with the microduplications, the reciprocal microdeletions are more likely to be penetrant and to be associated with non-specific major or minor dysmorphism. There are deletion positive ASD probands with a less severe phenotype than deletion negative ASD siblings underscoring the significant phenotypic heterogeneity.

Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: expanding the spectrum of Timothy syndrome.

Timothy syndrome (TS) is an autosomal dominant condition with the constellation of features including prolonged QT interval, hand and foot abnormalities, and mental retardation or autism. Splawski et al. [2004] previously described two phenotypes associated with TS distinguished by two unique and different mutations within the CACNA1C gene. We report on a newborn who presented with prolonged QT interval and associated polymorphic ventricular tachycardia, dysmorphic facial features, syndactyly of the hands and feet, and joint contractures, suggestive of TS. He developed a stroke, subsequent intractable seizures, and was found to have cortical blindness and later profound developmental delay. Initial targeted mutation analysis did not identify either of the previously described TS associated mutations; however, full gene sequencing detected a novel CACNA1C gene mutation (p.Ala1473Gly). The clinical and genetic findings in our case expand both the clinical and molecular knowledge of TS.

Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN)

Background Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN. Methods We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. Results We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease. Conclusions Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.

Ovarian failure and dilated cardiomyopathy due to a novel lamin mutation

Two unrelated young women presented with similar dysmorphic features including severe retrognathia, beaked nose, narrow chest, sloping shoulders, and an acrogeric appearance of the hands and feet. Neither had any evidence of skeletal myopathy, but both developed progressive dilated cardiomyopathy, both experienced premature ovarian failure, and both were found to have the same heterozygous novel missense mutation c.176T>G in exon 1 of the LMNA gene, resulting in a leucine to arginine change at codon 59 (Leu59Arg). Mutations in the LMNA gene cause a variety of disorders including dilated cardiomyopathy, muscular dystrophy, familial lipodystrophy, progeria, atypical progeroid syndromes, and mandibuloacral dysplasia. Genotype–phenotype correlation has been reported for some of these conditions. Our patients are the only ones known to have the specific mutation Leu59Arg and also share a set of features not entirely consistent with any of the laminopathies previously described. A previously reported patient with an adjacent mutation (Ala57Pro) had “atypical Werner syndrome” with dilated cardiomyopathy, hypogonadism, and sloping shoulders. While each of these clinical features does occur in other laminopathy syndromes, these patients form a phenotypic cluster distinct from other laminopathies and clinically overlapping with Malouf syndrome. LMNA sequencing should be considered for patients presenting with dilated cardiomyopathy and hypergonadotropic hypogonadism, including those previously diagnosed with Malouf syndrome.

A 5-Mb microdeletion at 6q16.1-q16.3 with SIM gene deletion and obesity†

Jia-Chi Wang,* Lesley Turner, Brenda Lomax, and Patrice Eydoux Cytogenetics Laboratory, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, Hamilton, Ontario, Canada Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Genetics, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada Cytogenetics Laboratory, Children’s and Women’s Health Centre of British Columbia, Vancouver, British Columbia, Canada

Molecular breakpoint mapping of 6q11‐q14 interstitial deletions in seven patients

Interstitial deletions involving 6q11‐q14 have been reported in less than 20 patients, with the breakpoints studied by G‐banding alone. We report on seven patients with 6q11‐q14 interstitial deletions of variable size. The breakpoints were studied by G‐banding, dual‐color BAC‐FISH and SNP array. The results showed the molecular breakpoints differed significantly from the ones obtained from G‐banding. The breakpoints studied by BAC‐FISH were consistent with the ones from SNP array. Some characteristics from this cohort are consistent with previous reports, but many typical features are lacking in our patients. The cardinal features of 6q11‐q14 interstitial deletions in this cohort include: umbilical hernia, hypotonia, short stature, characteristic facial features of upslanting palpebral fissures, low set and/or dysplastic ears, high arched palate, urinary tract anomalies, and skeletal/limb anomalies.

Deletion of the MC4R Gene in a 9-Year-Old Obese Boy

BACKGROUND The most common monogenic form of obesity is caused by mutations in the melanocortin 4 receptor (MC4R) gene. More than 150 mutations have been reported in the MC4R gene, the majority being point mutations. Most individuals with MC4R gene mutations have early-onset obesity, hyperphagia, and increased longitudinal growth. METHODS A 9-year-old Caucasian boy was referred to genetics for obesity, food-seeking behavior, and developmental delay. History and physical exam were not consistent with Prader Willi syndrome, but revealed several minor anomalies. Owing to significant obesity and hyperphagia, a Prader Willi syndrome methylation test and a microarray were requested. RESULTS Methlylation testing for Prader Willi syndrome was normal. Microarray analysis revealed two changes: (1) A 2.6-Mb deletion at chromosome 18q21.31 was identified and contained several OMIM genes, including the MC4R gene, and (2) an 0.87-Mb duplication at chromosome region 16p13.3 was found and contained one gene. Parental samples revealed that the boys father had the same deletion and duplication. This case appears to be the first with a deletion of 18q21.31 encompassing the MC4R gene presenting with features of hyperphagia and obesity. CONCLUSIONS Haploinsufficiency of the MC4R gene either through whole gene deletion or nonsense or missense mutations is associated with a significant risk of obesity. The case emphasizes both the role of the MC4R gene in obesity as well as the importance of looking for chromosomal microdeletions/duplications as a cause of obesity in children with minor anomalies or developmental delay.

Benefits and burdens of newborn screening: public understanding and decision-making

In this article we review the literature regarding the public understanding of the potential benefits and burdens of expanded newborn bloodspot screening. We draw attention to broadened notions of benefit that go beyond early identification of asymptomatic individuals and interventions to reduce morbidity or mortality, and include benefits gained by families through knowledge that may facilitate life choices, as well as gains generated by avoiding diagnostic delays. We also reflect on burdens such as increasing false-positive results and parental anxiety, together with risks of overdiagnosis when the natural history of a condition is poorly understood. We conclude that expanded notions of benefit and burden bring with them implications for parental consent and confidentiality and the secondary use of bloodspots.

Single-nucleotide substitution T to A in the polypyrimidine stretch at the splice acceptor site of intron 9 causes exon 10 skipping in the ACAT1 gene

β‐ketothiolase (T2, gene symbol ACAT1) deficiency is an autosomal recessive disorder, affecting isoleucine and ketone body metabolism. We encountered a patient (GK03) with T2 deficiency whose T2 mRNA level was <10% of the control, but in whom a previous routine cDNA analysis had failed to find any mutations. Genomic PCR‐direct sequencing showed homozygosity for c.941‐9T>A in the polypyrimidine stretch at the splice acceptor site of intron 9 of ACAT1. Initially, we regarded this variant as not being disease‐causing by a method of predicting the effect of splicing using in silico tools. However, based on other findings of exon 10 splicing, we eventually hypothesized that this mutation causes exon 10 skipping.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects’ cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.