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The Ulster Medical Journal logoLink to The Ulster Medical Journal
. 2010 Sep;79(3):137–145.

13th Meeting of the Irish Society of Human Genetics

PMCID: PMC3284721

PROGRAMME

10.00-10.55 – Registration/ Tea and Coffee

10.55 – Welcome

11.00-12.00 – Plenary I: Clinical Research- 5 Spoken Presentations

12.10-13.00 – Keynote address: “Advances in human genetics: what benefits for the patients?” Dr. Arnold Munnich, Hospital Necker-Enfants Malades and University Paris Descarte

13.00-14.00 – Lunch and Poster viewing

13.45-14.00 – Council meeting

14.00-15.30 – Plenary II: Basic Research- 6 Spoken Presentations

15.15-15.45 – Tea and coffee / Poster viewing

15.45-16.00 – Business meeting

16.10-17.00 – Keynote address: “A Singular view of the genome.”

– Prof. David Schwartz, University of Wisconsin-Madison.

17.00-17.45 – Wine Reception/ Presentation of Prizes / Meeting Close

Ulster Med J. 2010 Sep;79(3):137.

SO 1. An attenuated form of Morquio Disease seen in Northern Ireland

Fiona J Stewart 1, J Edmond Wraith 2, Karen Tylee 2, Alan Cooper 2

Morquio disease (mucopolysaccharidosis type IV) is an autosomal recessive lysosomal storage disorder causing predominantly skeletal manifestations. It is caused by a deficiency of galactose-6-sulphatase. In classical Morquio disease there is extreme short stature with height being between 90 and 120 cm. We have identified 10 individuals in Northern Ireland with an attenuated form of the disease. All were found to have glycosaminoglycans (GAG's) in their urine and reduced levels of galactose-6-sulphatase consistent with a diagnosis of MPS type IV Height ranged from 142 cm to 160cm (5* to 50th centile). 6/10 patients have had at least one major joint replaced with two having had 3 joints replaced. Our patients have also shown evidence of osteoporosis with decreased bone density being seen in all cases tested so far. Mutations in the GALNS gene have been characterised in all cases and include p.Il 13F, p.T312S and p.A241A. We believe this diagnosis should be considered in young people presenting with epiphyseal dysplasia and also in young adults presenting with joint problems requiring joint replacement surgery at an early age. Urine should be screened for GAG's in the first instance followed up by enzyme studies if keratan sulphate is detected.

Ulster Med J. 2010 Sep;79(3):137.

S02. Delineation of a recognisable phenotype of interstitial deletion 3 (q22.3q25.1) in a case with previously unreported truncus arteriosus

Gillian Rea a, Simon McCullough b, Susan S McNerlan b, Brian Craig c, Patrick J Morrison a

Interstitial deletions of chromosome 3q22.3-25.1 are very rare. We describe a case of a female infant with a de novo deletion. Chromosome analysis showed an interstitial deletion with a female karyotype 46,XX,del (3)(q23q25. l)dn. Subsequent array CGH demonstrated the breakpoints as 3q22.3q25.1. We identify an emerging clinical phenotype which includes, congenital heart disease, slow feeding, skeletal abnormalities and developmental delay. Characteristic facial dysmorphism includes ear anomalies, short neck, and small chin. Eye features include short palpebral fissures (blepharophimosis), ptosis and epicanthus inversus which are in keeping with the well delineated phenotype of BPES-blepharophimosis, ptosis and epicanthis inversus, a rare autosomal dominant disorder characterised by an eyelid malformation. FOXL2 (a putative forkhead transcription factor gene) has been identified as the causative gene, it is located on chromosome 3 at q22.3 In this case, array CGH demonstrated that the FOXL2 gene was not deleted, this was unexpected given the classical BPES features demonstrated. It has previously been shown that a small proportion of the molecular defects within a cohort of BPES patients have extragenic microdeletions, including those found downstream of FOXL2, as in our case. This may be the result of potential long-range czs-regulatory elements regulating FOXL2 expression.

Ulster Med J. 2010 Sep;79(3):137.

S03. Atypical 22qll deletion detected by multiplex ligation-dependent probe amplification (MLPA) in patients referred for Prader-Willi Syndrome (PWS) testing

Karen Meaney 1, Bronagh O'hIci 1, Sally Ann Lynch 1, David E Barton 1

PWS is caused by loss of a paternally-imprinted region on chromosome 15. It is characterized by hypotonia, short stature, hyperphagia, obesity, hypogonadism and mild mental retardation. We use MLPA to test for PWS. The MLPA kit contained a control probe located in the distal 22qll region (SNAP29 gene). We detected reduced dosage of this probe in two patients referred for PWS testing. One was obese and developmentally delayed at age 24 years, the other was a neonate with hypotonia and feeding difficulties. Follow-up investigation confirmed 22qll deletions in both cases. Chromosome 22qll contains multiple low-copy repeats (LCRs) which can mediate non-allelic homologous recombination, making the region susceptible to rearrangements. The most common rearrangement is a 3Mb deletion from LCR22-A to LCR22-D which is associated with 22ql 1 deletion syndrome. FISH analysis is frequently used to detect 22ql 1 anomalies. Recent publications have described novel 22qll deletions, some of which are distal to the commonly-deleted region and would not be detected during routine FISH analysis. Our results suggest an overlap exists in clinical features between PWS and 22ql 1 deletion syndrome, and demonstrate the importance of investigating MLPA control probe variations. Testing for atypical 22ql 1 rearrangements should be considered when PWS testing is negative.

Ulster Med J. 2010 Sep;79(3):137–138.

S04. Taking On Passing On: Long term experiences and needs in affected BRCA1/2 mutation carriers

Lisa Jeffers 1,2, Donna Fitzsimons 2, Eilis McCaughan 2, Patrick Morrison 1

Background: Research into the BRCA1/2 breast cancer susceptibility genes has yet to account for any long term psychosocial effects of genetic testing in gene carriers with a personal history of HBOC.

Aim of Study: This study was concerned with exploring the experience and needs of this group of women over a 2 year period.

Methodology: A grounded theory approach was taken using qualitative interviews (n= 49) and reflective diaries.

Analysis and Results: Taking On Passing On emerged as the basic social psychological process through which BRCA carriers with a personal history of HBOC respond to and resolve what is for them, a major concern – the passing on of a cancer gene to their offspring. Constant comparative analysis of the data traces the development of the process through the stages (1) Appraising Risk, (2) Formalising Risk (3) Minimising Risk and Maximising Survival and (4) Optimising Living. The theory contributes to the literature of transition theory, coping and adaption and psychological theories.

Implications for Clinical Practice: This prospective longitudinal study is relevant to clinical practice by contributing to our understanding of how women cope with learning their genetic status in the short and longer term and their ongoing needs once they leave a cancer genetic consultation.

Ulster Med J. 2010 Sep;79(3):138.

S05. Diagnostic Gene Screening of Cardiac Disorders in the N. Irish Population

SV Heggarty *, W Wright *, FJ Stewart *, PJ Hart *, P McKeown §, CA Graham *

The introduction of genetic diagnostic screening for cardiomyopathies and ion channelopathies in N. Ireland offers the prospect of early intervention and monitoring of asymptomatic patients at risk of developing fatal arrhythmias.

Hypertrophic Cardiomyopathy, a major cause of sudden death in young people is characterised by left ventricular hypertrophy in the absence of predisposing cardiovascular conditions. The sarcomere genes MYH7, MYBPC3, TNNT2 and TNNI3 of 52 patients were sequenced and pathogenic mutations identified in 29 patients (55.7%). In total 23 different mutations were detected, ∼60% of patients had mutations in MYH7 (5 clustered in exon 23, 2 double mutations), and ∼30% in MYBPC3 (1 compound heterozygote).

Long QT syndrome, a repolarization disorder of the heart, is identified by prolonged QT interval. It presents clinically in young people with episodes of syncope and potentially lethal torsades de pointes tachyarrhythmias. Screening of the KCNQ1, KCNH2, KCNE1 and KCNE2 genes has been carried out in 100 patients. 19 different mutations were identified in 54 patients, of which ∼65% are found in KCNQ1 (5 compound heterozygotes) and a further ∼30% in the KCNH2 gene.

Genetic screening in N. Ireland has proved an effective tool in detecting familial mutations for cascade screening of first degree relatives.

Ulster Med J. 2010 Sep;79(3):138.

S06. Functional and Neuropsychological Assessment of S100B as a susceptibility gene for Schizophrenia and Bipolar Disorder

Elif Dagdan 1,4, Derek W Morris 4, Matthew Hill 4, Matthias Rothermundt 5, Florian Kästner 5, Christa Hohoff 5, Jürgen Deckert 6, Christof von Eiff 7, Petra Krakowitzky 8, April Hargreaves 4, Emma Rose 4, Aiden P Corvin 4, Gary Donohoe 4, Michael Gill 4, Patrick McKeon 2,3, Siobhan Roche 1,2

The glial cell-derived neurotrophic factor, S100B, has been implicated in the pathology of bipolar affective disorder (BPAD) and schizophrenia (SZ). S100B protein levels are elevated in serum of patients with these disorders. We previously reported association of a S100B promoter SNP, rs3788266, with BPAD (P=0.0088). Here, we report that the disease-associated G allele of rs3788266 is associated with increased protein levels in serum of Irish BPAD probands (n=87), their first-degree relatives (n=67) and German controls (n=196). The G allele of rs3788266 is also associated with increased promoter activity in U373MG glioblastoma and in SH-SY5Y neuroblastoma cell lines as determined using the luciferase reporter system. Using an EMSA, the binding affinity of U373MG and SH-SY5Y protein complexes that bind to the S100B promoter were stronger on the G- compared to the A-allele promoter fragments. Finally, analysis of 433 SZ cases, 75 BPAD cases and 232 healthy controls identified a significant association between the G allele of rs3788266 and episodic memory, social cognition and verbal IQ. Overall, the data suggest that rs3788266 may represent a functional susceptibility variant that contributes to increased S100B serum levels observed in SZ and BPAD patients by increasing gene expression, which in turn impacts on cognitive performance.

Ulster Med J. 2010 Sep;79(3):138.

S07. Investigation of RNA-seq as a method of SNP detection

Emma M Quinn 1, Elaine M Kenny 1, Paul Cormican 1, Amy S Gates 1, Michael Gill 1, Aiden P Corvin 1, Derek W Morris 1

The development of Next Generation Sequencing offers the potential of new methods for mapping and quantifying transcriptomes. In particular, RNA sequencing (RNA-seq) has been used for measurement of transcript abundance, studying the diversity of splice isoforms and testing allelic influence on gene expression. Given that the majority of disease related SNPs are likely to be located in coding regions, we investigated RNA-seq as a method of identifying sequence variants (SNPs) in the transcribed regions of the genome. We performed RNA-seq using an Illumina Genome Analyzer II on lymphoblast cell line RNA samples from a trio of HapMap samples that have been whole-genome sequenced. We categorised genes and exons based on their overall X coverage and tested SNPs detected within these regions for concordance with the existing DNA sequence data. We found that at sufficient coverage (e.g. 20X), a high proportion of variants (80%) in a large number of genes/exons (∼2,400/20,744 for 1 x lane 80bp) can be accurately detected using this method. We also detected a number of novel SNPs that Mendelised within the trio, but were not reported from the published whole-genome sequencing data. Whole-genome re-sequencing is the most comprehensive method of variation detection but it is costly. We demonstrate that in addition to transcriptome analysis, RNA-seq can detect a very high proportion of sequence variation in expressed genes potentially making it a useful technique for detecting coding SNPs in disease tissue samples.

Ulster Med J. 2010 Sep;79(3):138–139.

S08. Gene-centric study identifies two novel genes, CLCN2 (a voltage-gated chloride channel) and KCNABl (a voltage-gated potassium channel) associated with blood pressure in two independent Irish populations

Nina McCarthy 1, Ciara Vangjeli 1, Gianpiero Cavalleri 1, Kevin Shianna 2, Norman Delanty 1, Eoin O'Brien 3, Brian Harvey 1, Alice Stanton 1

Hypertension is highly heritable. Loci identified by recent GWAS only explain a small proportion of total BP variation - identification of all genetic variants associated with BP will require complementary strategies. Here we report on a targeted candidate gene study with dense SNP coverage of multiple genes involved in electrolyte transport.

For the screening study 1860 SNPs in 81 genes were genotyped in healthy volunteers (n=358) with clinic BP measurements. For the replication study, 35 SNPs, in 21 genes, that were found to be associated with clinic systolic (SBP), diastolic (DBP) and/or pulse pressures (PP) in the screening study, were genotyped in a second independent population (n=380) for whom repeated ambulatory and clinic BP measurements were available. All association analyses were performed using an additive genetic model and adjusting for age and sex.

Table 1 summarizes the associations of the top two SNPs with BP - a synonymous SNP in a voltage-gated chloride channel gene (CLCN2) and an intronic SNP in a voltage-gated potassium channel gene (KCNAB1). These variants have not previously been implicated in the causation of hypertension. Our findings provide new insights into the pathophysiology of BP regulation, and may point to novel drug targets for hypertension treatment.

Table 1.

Differences in clinic and ambulatory BP in mmHg (meanP-value) per copy of minor allele for the two top signals in the screening and replication populations. Significant differences are in bold font

CLCN2SN9 KCNAB1SNP
Screening Population (MAF=0.21) Replication Population (MAF=0.21) Screening Population (MAF=0.04) Replication Population (MAF=0.05)
Clinic SBP 2.70.02 2.90.003 5.50.04 7.00.0002*
Daytime SBP 1.80.009 3.60.006
Night-time SBP 2.10.001* 3.50.005
Clinic DBP 2.10.009 1.00.1 0.30.8 3.00.01
Daytime DBP 2.00.00003* 1.30.2
Night-time DBP 2.00.00003* 0.90.3
Clinic PP 0.70.4 1.90.006 5.90.001 4.00.003
Daytime PP −0.20.6 2.30.009
Night-time PP 0.10.8 2.50.001*
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*

P-values i n the replication population which exceed the Bonferroni correction for multiple testing.

Ulster Med J. 2010 Sep;79(3):139.

S09. CNV detection using Parallel Targeting and Sequencing of Multiple Genomic Regions

Paul Cormican 1, Elizabeth A Heron 1, Elaine M Kenny 1, William P Gilks 1, Aiden P Corvin 1, Michael Gill 1, Derek W Morris 1

DNA copy number variation (CNV) has been recognized as an important source of genetic variation and in recent years extensive efforts have been undertaken to identify and correlate copy number variant genetic risk factors with disease phenotypes. Next Generation Sequencing (NGS) technologies have provided a potential new source of data for predicting CNVs and provide a feasible alternative to DNA microarrays for detecting such variants. In this study, we describe a sequencing-based strategy for high-throughput, cost-effective, targeted characterisation of structural variation including deletions, duplications and insertions. We have developed a method for CNV detection using read Depth Of Coverage (DOC) as a compliment to established Paired-End Mapping (PEM) strategies. We applied our algorithm to specifically targeted regions in HapMap individuals sequenced in-house, to varying levels of coverage. We tested the specificity of our method by examining the overlap between our predictions and regions of copy number variation which had previously been characterised in these HapMap individuals. Our method allows for the detection of copy number variable regions within targeted regions, quantification of copy number from the depth of read coverage as well as identification of genomic breakpoints at very high resolution.

Ulster Med J. 2010 Sep;79(3):139.

S10. Genome Wide Association Study of Non-synonymous Single Nucleotide Polymorphisms for even common diseases

Praveen Surendran 1,2, Alice Stanton 2, Denis Shields 1

Associations of several single nucleotide polymorphisms (SNPs) with common diseases were identified in a study conducted by Wellcome Trust Case Control Consortium. In this study the effects of genetic variations in 14,000 cases and 3000 controls and identified 24 independent associations with seven common diseases in European population. We hypothesize that there are more chances of finding associations of rare SNPs with diseases by refined analysis of non synonymous SNPs (nsSNPs) in genome wide association studies. In the present study, we analyzed the association of 12,660 nsSNPs using a case control study in the WTCCC population. We simulated the genotypes at 10,798 nsSNP loci studied by the Stage 2 HapMap project using the genotype information from WTCCC for all 14,000 individuals studied for seven diseases and in 3000 controls. Subsequent case control association of 10,798 imputed nsSNPs and 1,862 genotyped nsSNPs was performed using an additive model and genotype model in a frequentist and bayesian framework. We have identified 4 nsSNPs associated with CD, 8 with RA, 5 with T1D and 1 with T2D. In total, 18 new associations with the seven diseases (p < 5 × 106) studied by WTCCC. We also developed a pipeline which summarizes quality control measures which should be considered to minimize false associations in genome wide association studies.

Ulster Med J. 2010 Sep;79(3):139.

S11. Screening of the NTD-associated MTHFD1L Gene Polymorphisms in an Irish Cleft Cohort

S Minguzzi 1, A Molloy 2, P Kirke 3, J Mills 4, J Scott 5, J Troendle 4, F Pangilinan 6, L Brody 6, A Parle-McDermott 1

Human gene MTHFD1L (methylenetetrahydrofolate dehydrogenase 1-like) produces 2 alternatively spliced mRNA transcripts; the short form lacking synthetase activity.1 Previously, we demonstrated that polymorphisms adjacent to MTHFD1L alternative exon (8a) were associated with case risk of neural tube defects (NTDs) in the Irish population.2 Similarly to NTDs; there is a link between folic acid and cleft prevention. In this study, we genotyped MTHFD1L DIP (Deletion/Insertion Polymorphism) rs3832406 and SNP (Single Nucleotide Polymorphism) rsl7080476 in 981 Irish case-parent trios affected by clefting. Both assays were genotyped using Melting Curve analysis with HybProbes on a Roche LightCycler480® instrument. As this method is primarily designed for SNP genotyping where there are just 2 alleles, it was necessary to develop a novel assay in order to genotype the triallelic DIP rs3832406 polymorphism. Tests for association will be assessed by logistic regression and transmission disequilibrium test (TDT).

We demonstrate that Melting Curve analysis can be employed to successfully genotype challenging polymorphisms such as DIP rs3832406 and is a viable alternative to capillary electrophoresis. Results of this association analysis will be presented.

References

  • 1.Prassannan P, et al. J Biol Chem. 2003;278:43178–43187. doi: 10.1074/jbc.M304319200. Parle-McDermott A, et al, 2009. Hum Mut 30 (12): 1650-1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
Ulster Med J. 2010 Sep;79(3):139.

P01. Variable Phenotypic Consequences of a Terminal Deletion of Chromosome 8p

A Carey 1, S A Lynch 1, A-M Hegarty 1, D R Betts 1

Chromosomal abnormalities are a frequent underlying reason for developmental delay in children. We report a family in which a brother and sister (aged 14 and 12 respectively) were referred for cytogenetic analysis due to similar phenotypes of moderate developmental delay and very mild dysmorphic features. Both siblings had an apparently identical terminal deletion of chromosome 8p, extending into 8p23. The presence of this event in both children would indicate parental inheritance, which in most instances is due to one parent being a balanced translocation carrier. However, on this occasion the children's mother also had an apparently identical 8p deletion. To support this, subtelomere FISH analysis with chromosome 8 probes was performed and demonstrated loss of the 8p subtelomere in all individuals, thereby excluding a semicryptic translocation in the mother. The mother displayed no obvious phenotypic anomalies, but did report a non-clinical VSD diagnosed when she was a child. This family illustrates the challenges faced in predicting phenotypic consequences of rare chromosomal abnormalities that are not directly associated with a well defined syndrome. Further, future genetic counselling of the children when they reach reproductive age is difficult due to the unpredictability of the potential implications of this 8p deletion.

Ulster Med J. 2010 Sep;79(3):139–140.

P02. Optimisation and evaluation of MLPA for non-syndromic X-linked mental retardation

A Beckett 1, G Smith 1, S McCullough 1, M Humphreys 1, T Dabir 1, S McKee 1, A Magee 1, F Stewatr 1, V McConnell 1

Mental retardation (MR) is characterised by a significant impairment of cognitive and adaptive functioning and is estimated to have an incidence of approximately 1-3% in the population.

X-linked forms of MR can be divided into syndromic and non-syndromic forms. Syndromic X-linked MR is present in combination with a specific pattern of physical, neurologic and/or metabolic abnormalities. Non-syndromic X-linked MR describes a condition which segregates in an X-linked manner in which male patients have no consistent phenotype manifestations other than MR.

The aim of the project was to optimise and evaluate multiplex ligation dependent probe amplification (MLPA) as a technique for identifying patients with non-syndromic X-linked MR. The kit used, SALSA MLPA kit P106 MRX, obtained from MRC Holland, can detect copy number changes of 16 genes on the X chromosome that have been implicated in non-syndromic X-linked MR (RPS6KA3, ARX, IL1RAPL1, TSPAN7, PQBP1, HUWE1, OPHN1, ACSL4, PAK3, DCX, AGTR2, ARHGEF6, FMR1, AFF2 (FMR2), SLC6A8 and GDI1).

We have evaluated this MLPA test in a group of 52 patients referred with a broad phenotype of developmental delay and/or learning disability, together with a strong X-linked pattern of inheritance. No abnormalities were detected in the patients' samples. As a result of this project, it can be concluded that this test is not a practical technique to be implemented as a routine diagnostic screening method within the Belfast Genetics laboratory at the present time. A more appropriate first line test for patients in this referral category would be array comparative genomic hybridisation (aCGH).

Ulster Med J. 2010 Sep;79(3):140.

P03. A novel mutation causing retinitis pigmentosa (RP) identified in the cyclic nucleotide gated channel beta 1 (CNGB1) gene using massively parallel sequencing

DA Simpson 1, L Harwood 1, GR Clark 1, S Alexander 1, G Silvestri 1, C Willoughby 1

Introduction: Screening the >40 genes implicated in RP is a challenge for molecular diagnostic screening. Targeted massively parallel DNA sequencing was used to develop a potential solution.

Methods: A custom sequence capture array was designed to target the coding regions of all known RP genes and used to enrich these sequences from five patients and a pool of 360 controls. Enriched DNA was subjected to sequencing (Genome Analyzer) and variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls.

Results: Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. Common variants were identified in the pooled control sample. A novel homozygous missense mutation (c.2957A>T; p.N986I) in the CNGB1 gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations.

Conclusions: Sequence capture of a disease-specific cohort and subsequent high-throughput DNA sequencing can be used as a cost-effective genetic diagnostic tool, exemplified by the detection of a mutation in CNGB1, a rare cause of recessive RP.

Ulster Med J. 2010 Sep;79(3):140.

P04. Infantile onset of complex I deficiency due to a m. 10191T>C mutation in the mtND3 gene

Deirdre E Donnelly 1, Patrick J Morrison 1

Complex I deficiency is a rare mitochondrial disorder. Mutations have been identified in at least nine nuclear genes and eight mutations have been found in mitochondrial DNA. We report a patient with a rare mutation, m.lO191T>C, inthemtND3 gene who presented with macrocephaly, global developmental delay and seizures in infancy, and highlight the clinical phenotype of this rare disorder.

Ulster Med J. 2010 Sep;79(3):140.

P05. The prevalence of thanatophoric dysplasia and lethal osteogenesis imperfecta type II in Northern Ireland – a complete population study

Deirdre E Donnelly, Vivienne McConnell, Anne Paterson *, J Morrison Patrick

The minimum prevalence of lethal achondroplasia, osteogenesis imperfecta type II and thanatophoric dysplasia were derived following detailed case note review of all perinatal lethal skeletal dysplasias in Northern Ireland over a 12-year period. Multiple sources of ascertainment, including genetic notes, radiological reports and post mortem findings, were used. 39 cases were identified. Thanatophoric dysplasia was the commonest diagnosis made (22 children), followed by osteogenesis imperfecta type II (four children) and achondroplasia (two children). Eleven other diagnoses each occurred once in the 12-year period. The minimum prevalence range, per live births, of each of the common skeletal dysplasias in Northern Ireland has been calculated; thanatophoric dysplasia 0.81/10,000 and osteogenesis imperfecta type II 0.15/10,000. The prevalence range for thanatophoric dysplasia is much higher than reported in previous studies. We discuss reasons for the prevalence figures obtained.

Ulster Med J. 2010 Sep;79(3):140.

P06. Phenotypic variability in a three-generation Northern Irish family with Sotos Syndrome

Deirdre E Donnelly 1, Peter Turnpenny 1, Vivienne PM McConnell 1

Sotos syndrome is a relatively common overgrowth disorder, following autosomal dominant inheritance, caused by mutations and deletions in the nuclear receptor Set domain containing protein-1, NSD1 gene. In general almost all affected individuals have advanced bone age, macrocephaly, characteristic facial gestalt and learning difficulties. Other features include scoliosis, seizures, cardiac defects and genitourinary anomalies. Tumours are a rare occurrence. Genotype-phenotype correlations are unclear, though those with a deletion appear to have more severe mental retardation. Full penetrance is seen, although familial Sotos syndrome is extremely rare. The low vertical transmission rate, (not fully explained by cognitive impairment), is of great importance, particularly for mildly affected patients. We report a 3-generation pedigree with 7 affected individuals shown to harbour the NSD1 missense mutation c. 6115C>T. To our knowledge this is the largest Sotos family to be reported. The observed phenotype is extremely variable, both physically and cognitively, thus highlighting the lack of precise genotype-phenotype correlations in Sotos syndrome, which previous extensive studies have highlighted. Our family provides further evidence that NSD1 mutational analysis provides little prognostic information as many individuals with the same mutation have very different phenotypes. NSD1 gene mutations should be considered in a wider phenotypic range.

Ulster Med J. 2010 Sep;79(3):140.

P07. GLA mutation screening identified 17 cases of Fabry Disease

ED Beattie *, CA Graham *, FJ Stewart *

Fabry disease, an X-linked recessive disorder caused by α-galactosidase A deficiency, is a multi-system disease with variable age-of-onset. A significant proportion of female carriers also present with symptoms, particularly later in life.

The Northern Ireland Regional Genetics Centre offers a comprehensive screening service available through the UKGTN utilising sequencing and MLPA. Screening has been performed on 57 probands (21 male and 36 female) and mutations detected in 13 males (62%) and 4 females (11%). This resulted in cascade screening of 56 patients and the identification of 28 mutation carriers.

Fourteen mutations, all but two of which were private, were detected consisting of 6 missense, 3 nonsense, 2 splice site, 2 deletions and deletion of exon 1. Five mutations appear novel and of these, a nonsense mutation and deletion were considered pathogenic. The other missense mutations were detected in 5 patients all presenting with cardiac problems. Although the pathogenicity of one variant is uncertain, the others are thought to be consistent with a cardiac variant presentation.

In males with suspected Fabry disease, the pick-up rate is high but our experience suggests that Fabry disease is not a major contributor of disease in females with no family history of the condition.

Ulster Med J. 2010 Sep;79(3):140.

P08. A case of Menkes disease due to a previously unreported variant

Gillian Rea a, Deirdre E Donnelly a, Lisbeth Birk Møller b, Joanne Hughes c, Patrick J Morrison a

Menkes disease and occipital horn syndrome (OHS) are rare disorders of copper transport. Inheritance is in an X-linked recessive manner. Approximately one-third of affected males have no family history of Menkes disease/OHS. Infants with classic Menkes disease appear healthy until the age of two to three months, when loss of developmental milestones, hypotonia, seizures, and failure to thrive occur. There are characteristic changes of the hair (short, sparse, coarse, twisted, often lightly pigmented-pili torti).

ATP7A is the only gene known to be associated with Menkes disease and OHS. A multiplex protocol of targeted mutation analysis (MLPA), mutation scanning, and sequence analysis detects mutations in more than 95% of affected individuals. We describe a case of Menkes disease where a previously unreported variant was identified in both the affected child and their mother. This variant, c.2781G>A (p.K927K) does not change the amino acid but might affect the splicing of exon 13. RNA analysis from the index case and fibroblast culture were undertaken to determine if the variant was disease causing.

Ulster Med J. 2010 Sep;79(3):141.

P09. X marks the spot: Duchenne muscular dystrophy (DMD) presenting in a female child

G Rea a, S Tirupathi b, B Herron c, E Healy c, P Woods d, D Beattie d, F Stewart a

The mechanism of chromosomal rearrangements involving translocations between the X chromosome and an autosome, one breakpoint involving the Dystrophin gene and resulting in a DMD phenotype in a female, have been recognised since the late 1970's.

Two such cases have been known to the Northern Ireland Regional Genetics Service throughout its history. We describe the presentation and clinical features of the most recent case, a 4 year old girl who was investigated for proximal muscle weakness. At presentation she had bilateral calve hypertrophy, a broad based gait and positive Gower's sign, early contractures were developing around her Achilles tendons. CK was 20-25,000 and a muscle biopsy showed a myopathic picture with fibrosis consistent with dystrophy. There was some dystrophin present but it was absent or substantially diminished in many fibres.

Chromosome analysis revealed a de novo balanced translocation; 46, X, t (X;17) (p21.2;qll.2). The breakpoint is at the site of the dystrophin gene. We also review the alternative mechanisms by which females may present with a DMD phenotype (skewed X-inactivation or females with a disease causing DMD mutation and a complete or partial absence of their second X chromosome (45X or uniparental disomy –UPD).

Ulster Med J. 2010 Sep;79(3):141.

P10. A diagnosis of Tuberous Sclerosis Complex (TSC) in teenage years: the importance of follow-up

H Crawford 1, S McKee 1

We present a twenty-year old female patient with TSC who was first referred to the genetic service in 2005 at the age of fourteen years by the dermatology service on the basis of facial rash. This patient was asymptomatic for the major signs of TSC such as learning difficulty and seizures. At the Tuberous Sclerosis (TS) clinic a diagnosis of TSC was confirmed. Subsequent mutation analysis identified a deletion in exons 28-31 of the TSC2 gene (probable mosaic). This patient found the diagnosis of TSC difficult to cope with. She failed to attend appointments at the TS clinic over a three-year period, and failed to attend for renal ultrasound screening as recommended in the clinical guidelines. She attended for renal ultrasound screening for the first time in 2010 when a 5cm mixed density nodule was identified in the left kidney. There was also a second moderately sized 2.5cm angiomyolipoma in the same kidney. Further imaging by CT scan has been arranged to further evaluate these lesions. This case highlights the importance of encouraging patients with TSC at all stages in life to attend for recommended screening, and identifies the issue and difficulties of non-compliance in teenage years.

Ulster Med J. 2010 Sep;79(3):141.

P11. A Familial t(15;22)(q13;q11.1), Implications for Prenatal Genetic Testing

J McDaid 1, AM Hegarty 1, J Turner 1, G Clarke 1, T Morris 1, B O'hIci 1, K Meaney 1, A Green 1, DR Betts 1

Pregnancy in a familial balanced translocation carrier can pose a challenge to a genetics testing laboratory, particularly when the rearrangement is G-band subtle and involves chromosome 15. We report a t(15;22)(q13;q11.1) familial translocation that could appear to be subtle at lower banding resolutions. The mother, a phenotypically normal carrier of the translocation, reported a family history of a Prader-Willi like syndrome. FISH analyses were undertaken to define the translocation further and demonstrated that 15q breakpoint was distal to SNRPN, while the 22q breakpoint was in the alpha-satellite region of 22q11. Therefore the derivative chromosome 15 contained both SNRPN and the 22q11.2 region associated with Di George syndrome. At gestational week 11 a CVS biopsy was taken and sent to us for cytogenetic, MLPA and UPD studies. Due to the prior FISH testing, cytogenetic analysis could confidently define the foetal karyotype as: 46,XY,+der(15)t(15;22)(q13;q11.1),-22. This karyotype therefore demonstrates partial trisomy of 15pter-15q13 and insignificant monosomy of 22pter-22q 11.1, with the MLPA and UPD studies also consistent with this finding. These results illustrate the importance of prior planning for prenatal testing of an individual with a family history of a balanced chromosomal rearrangement and the identification of the appropriate tests and markers.

Ulster Med J. 2010 Sep;79(3):141.

P12. 17q21.31 microdeletion syndrome detected by Multiplex Ligation-dependent Probe Amplification (MLPA)

J Jones 1, SJ McCullough 1, F J Stewart 1, G Smith 1

High resolution aCGH has identified new genomic disorders in individuals with mental retardation and developmental delay. The 17q21.31 microdeletion syndrome is one of these disorders and is associated with a 500-650kb deletion encompassing the CRHR1 gene and the MAPT gene. The deletion mechanism is most likely non-allelic homologous recombination (NAHR) facilitated by a common 900kb inversion polymorphism.

Clinical features of the syndrome include developmental delay, hypotonia, facial dysmorphism, friendly/amiable behaviour, epilepsy, heart defects and kidney/urologic anomalies.

Our male patient, now 10 years old, first presented as a newborn with marked hypotonia, asymmetric IUGR and hypogonadism. He has thick curly hair, a short neck, a bulbous nasal tip, low set rotated ears, prominent forehead, irregular teeth, very hyperextensible joints and learning difficulties.

Microdeletion syndrome investigations using the MRC Holland MLPA kit, P245-A2, identified a microdeletion of the 17q21.31 region. This was further characterised using the MRC Holland MLPA kit, P371-A1. All 8 probes spanning the CRHR1 and MAPT genes were deleted.

Ulster Med J. 2010 Sep;79(3):141.

P13. A qualitative prospective analysis of cancer genetic referrals to the Northern Ireland Regional Genetics Service (NIRGS)

Lisa Bradley 1, Vivienne McConnell 1

The NIRGS is a tertiary referral speciality, receiving all cancer genetic referrals within Northern Ireland. It is recognized that some of the referrals to the NIRGS are inappropriate or lack specific details in order to either determine appropriateness or to have a more structured and efficient approach to facilitate the patient care pathway. The broad aim of this audit was therefore to identify the a) appropriateness (according to departmental and NICAM guidelines) and b) deficits, in cancer genetic referrals, in order to improve the patient care pathway and departmental workload. This had not been formerly assessed previously to determine the extent of appropriateness or to develop a specific regional cancer genetics referral proforma. The results of a qualitative prospective analysis of the first 100 cancer genetic referral letters received from the 1th April 2009 are presented. These included the findings that over 50% of referral letters had no documented contact patient telephone numbers or details of other related family members who had previously attended the Genetic Service with 33% omitting the age of diagnosis of affected relatives or probands. Based on these results, a cancer genetics referral proforma was developed, which is currently being piloted prior to implementation with re-audit planned in the future.

Ulster Med J. 2010 Sep;79(3):141–142.

P14. A diagnosis of Bardet-Biedl Syndrome despite unusual ophthalmic presentation

Lisa Bradley 1, Colin Willoughby 1, Vivienne McConnell 1

The 13 year old male proband is the third child of non-consanguineous parents, born full term after an uneventful pregnancy. He was initially referred to the Northern Ireland Regional Genetic Service (NIRGS) with obesity, moderate learning difficulties, behavioural problems and dysmorphism. Additionally, there was a history of chronic, severe glue ear requiring repeated surgical intervention. Subsequent ophthalmic evaluation reported only ‘some slight retinal pigment epithelial mottling’ at approximately 9yrs of age but fundus and macular examinations were normal, as were electroretinograms on two occasions.

A renal ultrasound, skeletal survey including bone age and initial genetic investigations were all normal. Despite clinically not meeting the diagnostic criteria of Bardet-Biedl Syndrome (BBS) suggested by Beales et al, 2001, subsequent BBS analysis was completed. This testing detected the proband to be homozygous for the missense mutation M390R in the BBS1 gene, confirming a BBS diagnosis. A review of this case and the literature is presented. This case highlights the need to consider the possibility of BBS even if suggested diagnostic criteria are not met.

Ulster Med J. 2010 Sep;79(3):142.

P15. Cat Eye Syndrome: The Northern Ireland experience and review of the literature

Lisa Bradley 1, Katherine Murtagh 1, Vivienne McConnell 1

In 1965 Schmid in Zurich and Fraccaro in Pavia first reported on the association of coloboma and anal atresia with a small extra chromosome and proposed the term ‘Cat Eye Syndrome (CES)’. It is now known that CES (estimated incidence between 1:50 000 and 1:150 000) is typically associated with a small supernumerary bisatellited marker chromosome (inv dup 22pter-22ql 1.2), resulting in four copies of this region. CES is known to exhibit extensive phenotypic variability (ranging from near normal to severe malformations). The cardinal features include coloboma, pre-auricular tags and/or pits, anal and cardiac defects and renal malformations.

A retrospective review of all CES cases presenting to the Northern Ireland Regional Genetics Service was undertaken. Cases were ascertained through chart, database and laboratory records with clinical features, phenotypic variability and genetic results being the factors assessed. A total of nine cases were identified, two of which were inherited, and 2 mosaic. Interesting only 1 had coloboma the feature from which this syndrome derived its name. The majority of our cases demonstrated developmental delay which is not one of the regarded cardinal features. Further delineation of our cohort with respect to clinical features, genetic aetiology and review of the literature will be presented.

Ulster Med J. 2010 Sep;79(3):142.

P16. Familial Subtelomere Duplications or Deletions

L Ekstrom 1, M Mullarkey 1, T Morris 1, S A Lynch 1, A Green 1, DR Betts 1

Duplications or deletions of subtelomere regions are detectable by either FISH or MLPA methods. These tests have been widely reported as valuable in detecting subtle chromosomal aberrations that have phenotypic consequences. Nevertheless familial duplications or deletions, the most common of which involve lOq and 4q, also exist. We focus on two cases that were subsequently referred to our laboratory following MLPA subtelomere investigations in an amniotic fluid sample, sent to exclude cystic fibrosis, and a boy with developmental delay. MLPA investigations performed by another institute had reported a duplication of 15q and 7q respectively. G-banding of the amniotic fluid sample had been performed at the NCMG and no visible abnormality seen. Subsequent parental FISH analysis showed the phenotypically normal father to have an enhanced 15q signal, consistent with a MLPA duplication result. Without family studies in the second case the subtelomere duplication of 7q (G-band was normal) could be have been interpreted as the explanation for this boy's developmental delay. However, again FISH showed inheritance from a phenotypically normal father. These and other cases illustrate potential serious consequences of reporting subtelomere anomalies in the absence of other abnormal test results or family studies.

Ulster Med J. 2010 Sep;79(3):142.

P17. A Patient with AML and MDS-related changes, showing a complex karyotype including excessive telomere association

M Humphreys 1, Amy Logan 1, Peter McGrattan 1, G Marron 2

A 74 year old man presented initaially in April 2008 with mild asymptomatic anaemia. Bone marrow (BM) cytogenetic analysis showed a very complex hypodiploid karyotype. BM morphology at this time showed trilineage dysplasia with no excess blasts.

The patient showed progressive fall in platelets during the next nine months and developed fever, epistaxis and severe lethargy. BM morphology showed transformation to AML.

Follow up BM cytogenetics detected a near triploid clone with some of the abnormalities detected previously. However, on this occasion most of the abnormal metaphases included pairs of chromosomes involved in telomere associations. Twenty different pairings were seen, one of which, between 15p and 22p, occurred in three cells.

Telomere association is relatively rare but has been reported in several different solid tumour types, in lymphoproliferative disorders and in ataxia telangiectasia. To our knowledge this phenomenon has not been previously reported in myeloid malignancy. It's biologic and clinical significance is unclear.

Ulster Med J. 2010 Sep;79(3):142.

P18. Uptake of Huntington disease predictive testing in a complete population and calculation of the prevalence

Patrick J Morrison 1, Siobhan Harding-Lester 1, Aoife Bradley 1

Introduction: Estimates of numbers of at risk patients availing of Huntington disease (HD) predictive testing are inaccurate as most patients who do not want testing simply do not attend clinics.

Methods: We used the Northern Ireland HD register to analyse the number of prospectively recorded predictive tests over a 20 year period and by calculating the prevalence and counting the number of at risk cases, have estimated the total uptake in a defined population.

Results: 212 patients completed predictive testing between 1990 and 2009. 92 (43%) received mutation positive results and 119 (56%) mutation negative. There was one intermediate allele result. There was no significant gender difference. 180 affected cases confirmed by molecular genetic testing were alive on the first of January 2001. The uptake of predictive testing in the entire HD 50% at risk population was calculated by three methods giving a range of 18.3-22.1%. Applying correction factors, the uptake after 10 years of testing was 29.2% and after 20 years was 22.1%. The prevalence of affected HD cases was 10.6 / 100,000 in 2001.

Conclusions: Total uptake of predictive testing has not previously been calculated and suggests that over two thirds of at risk patients do not come forward for testing until symptomatic. Presymptomatic testing for this late onset condition with no present treatment, and limited management options, still presents challenges for families.

Ulster Med J. 2010 Sep;79(3):142.

P19. Multiplex MassARRAY spectrometry (iPLEX™) testing for Familial Hypercholesterolaemia

PJ Hart *, SV Heggarty *, WT Wright *, DP Nicholls †, CA Graham *

An iPLEX™ assay testing for 55 LDLR mutations, ApoB p.R3527Q , and PCSK9 p.D374Y was developed in Belfast in 2007 as an inexpensive and rapid first-line test for Familial Hypercholesterolaemia.

A total of 864 patients with elevated cholesterol attending lipid clinics underwent FH testing via this assay. These include referrals from laboratories throughout the UK and from a variety of ethnic/genetic backgrounds. Mutations were identified in 117 (14%) of these patients and subsequently confirmed by direct sequencing. Following clinical review, a number of iPLEX-negative patients underwent fluorescent sequence analysis of the promoter and coding sequence (including exon-intron boundaries) of the LDLR gene, and of PCSK9 (exon 7) and ApoB (exon 26), and MLPA analysis. Of the 201 patients to date who have completed this, mutations have been identified in 62 (31%).

iPLEX™ analysis identified 43 different mutations in a total of 117 patients. The four most common mutations identified were ApoB p.R3527Q (12 patients), p.ElOlK (11 patients), c.2292delA and p.C231X (8 patients each). Neither of these last two mutations would have been detected by the Tepnel Elucigene assay. Tepnel analysis would have identified 18 different mutations in 73 patients (8.4%). iPLEX™ analysis therefore remains a cost-effective first-line screen for FH.

Ulster Med J. 2010 Sep;79(3):142–143.

P20. Array CGH: Comparison of two commercially available platforms for the investigation of childhood developmental delay

SE McNerlan 1, P McGrattan 1, S Heggarty 1, P Erwin 2, M Humphreys 1

Array comparative genomic hybridisation is becoming an increasingly important and useful tool in the cytogenetic investigation of children with learning difficulties, dysmorphism and developmental delay. Currently there are a number of array platforms commercially available. We carried out a pilot study to compare the performance of 2 systems: a 4x44k oligonucleotide array (Agilent) and the Human CytoSNP 12 bead chip system (Illumina). Patients were children with apparently normal karyotypes, where previously performed microdeletion and subtelomere MLPA gave no diagnosis. The platforms were used to determine gains/losses of genetic material and also, in the case of the SNP arrays, regions of copy number neutral loss of heterozygosity. Forty-six patients were included in the study, alongside 16 abnormal controls, with 37 patients and controls run in parallel on both systems. Both platforms correctly identified all known abnormalities in 15 of the controls, however, one control with a small subtelomeric anomaly, run only on the oligonucleotide array, was not readily called. Both systems detected potentially clinically significant abnormalities in 5 patients (8%). Abnormalities ranged in size from approximately 0.4Mb to 2.5Mb and included a del(14q), del(3p) and del(16p). Both platforms were therefore found to be robust and showed high level of concordance.

Ulster Med J. 2010 Sep;79(3):143.

P21. Non-multifactorial Neural Tube Defects in Northern Ireland

Tabib Dabir 1, Fiona Stewart 1

Neural Tube Defects (NTDs) are generally considered to have multifactorial inheritance. However it has been reported that 2-16% of isolated NTDs and up to 25% of NTDs with other congenital malformations have a chromosomal abnormality. More than 50 %of spontaneous abortions with NTDs have abnormal karyotype. Syndromic NTDs are associated with other congenital malformations and show normal karyotype.

Our recent study of NTDs in Northern Ireland (NI) for the period of 2000-04 identified 125 cases with prevalence of 1 in 1000. Thirteen NTDs cases (10%) had multiple congenital malformations. Chromosome analysis was done in 10 and a cytogenetic abnormality was identified in 4 cases (40%) in this group. Chromosome analysis was done in total 24 cases of NTDs (12 amniocentesis and 12 postnatal) during the study period and abnormal karyotype was reported in 4 cases (17%). Non-multifactorial NTDs are relatively rare. However it is important to be aware of this group as the recurrence risk maybe higher than the common multifactorial NTDs. Chromosome analysis and detailed clinical or post-mortem examination should be done in all cases of NTDs. This may help in identifying causative loci/ genes and also to provide accurate genetic counselling to family members.

Ulster Med J. 2010 Sep;79(3):143.

P22. Thirty-year trends in birth prevalence and antenatal diagnosis of Trisomy 18 and Trisomy 13 in Northern Ireland

Tabib Dabir 1, David G Grier 2, Aoife Bradley 1, Geoff Smith 1, Alex Magee 1, Patrick J Morrison

Trisomy 18 and Trisomy 13 are the second and third most common trisomies in live-born babies. The aim of this study were to determine the trends in birth prevalence and the antenatal diagnosis of trisomies 13 and 18 in Northern Ireland during a thirty year period by retrospective examination of clinical records. All trisomy 13 and 18 diagnoses during the period from 1979 to 2008 were identified from the Northern Ireland Regional Cytogenetic Laboratory and demographic details obtained. The prevalence at birth of live-born babies was 1/6508 for trisomy 18 and 1/14244 for trisomy 13 for the study period. Poisson regression analysis was used to compare the birth prevalence in three periods (1979-98, 1999-2003, 2004-08) and in the five maternal age categories. Increased maternal age (35 years and above) was noted to be an important factor in both trisomies. There was no significant change in the prevalence of trisomy 13 over the period of thirty year. However there was a significant rise in trisomy 18 cases for the last five year study period (2004-08) with the live birth prevalence of 1 /3105. This rise in prevalence was not totally explained by maternal age or termination of affected pregnancy (Chi2 = 7.39, df =8, P =0.50) and is likely to be real. Similar trend was not noted in trisomy 13. There has been an increased in prenatal diagnosis of each trisomy and it is mainly related to abnormal scan findings.

Ulster Med J. 2010 Sep;79(3):143.

P23. Management and Screening of VHL- Who's Responsibility is it?

Rachel Hardy 1, Tabib Dabir 1

Von Hippel-Lindau (VHL) is an autosomal dominant disorder characterised by development of cancerous and noncancerous tumours of various organs. VHL patients need regular surveillance by various medical professionals. Early detection and intervention through surveillance prevents or minimizes deficits such as hearing loss, vision loss and neurologic symptoms related to these tumours and there are recognised recommended surveillance protocols for VHL and its clinical management. Due to the complexity of this condition many specialist clinics have been established in the UK, coordinated and managed by the local genetics department.

This audit aimed to assess the current screening arrangements for VHL patients in Northern Ireland by conducting a patient satisfaction survey. The objective was to assess the need for centralisation and co-ordination of their care by a regional multidisciplinary VHL clinic. Patients known to our service were identified and sent a postal questionnaire comprising ten questions related to current surveillance and their feedback. In view of limited numbers this was followed by a telephone call to maximise the response.

The results of this audit support the need for co-ordinated approach to VHL related surveillance and advocates the establishment of a regional VHL clinic led by the clinical genetics department.

Ulster Med J. 2010 Sep;79(3):143.

P24. Hyperlipidemia, Hypertriglyceridemia contributing Premature Cardiovascular Disease in Type II Diabetes (T2D) Patients: The Prospective Hospital Based Study

V Bhupeshkumar 1, Vijaya Kumar 2, Sudhir Naik 3, Subash Sadhnani 4

Cardiovascular disease (CVD) is a leading cause of death and disability worldwide, with genetic and environmental predisposition. Majority of Cardiovascular disease occur in individuals >65 year old, but is being increasingly seen in individuals with age <50 years. In the present study APOC3 (C3238G), APOC3 promoter (C482T) and APOA5 (T1131C) genes involved in regulation of lipid metabolism and has recently been implicated in the pathogenesis of CVD in T2D patients. We investigated the effects of common variants of these genes on T2D patients as well as the association with CVD. Genomic DNA is extracted from blood samples obtained of 120 T2D subjects and 150 healthy volunteers following both inclusive and exclusive criteria. DNA samples were genotyped for C3238G, C482T variants using PCR-RFLP assay and then DNA samples followed by genotyping T1131C variant using ARMS-PCR assay. The 3238C>G, 482C>T SNP of APOCIII (S2 and TT allele) tend to have high plasma triglyceride concentrations was shown a risk factor in T2D subjects when compared with healthy volunteers. The rare allele 1131T>C (CC allele) is also associated with elevated lipids levels. The Haplotypes analysis performed for APOAV and APOCIII SNPs indicated significant differences (P<0.001) in distribution. Most significantly, a risk haplotype S2/T/C was obtained (P<0.001) and was associated with increased risk of CVD.

Ulster Med J. 2010 Sep;79(3):143–144.

P25. Association of Variants in Candidate Genes Influencing Autonomic Nervous System Functionality with Blood Pressure Level

Ciara Vangjeli 1, Nina McCarthy 1, Gianpiero Cavalleri 1, Kevin Shianna 2, Norman Delanty 1, Eoin O'Brien 3, Alice V Stanton 1

Genomewide association studies have identified 13 novel loci associated with blood pressure (BP). However, only a small proportion of total BP variation is explained by these findings. Here we report on a candidate gene study with dense SNP coverage of multiple genes involved in the autonomic nervous system (ANS), namely neurotransmitter receptors, metabolisers, and transporters. Using the Illumina GoldenGate platform, we genotyped 2364 SNPs in 168 genes in 358 healthy bank employees who had undergone clinic BP measurements (screening population, SP). Only the 58 SNPs in 33 genes, found to be associated with systolic, diastolic or pulse pressure within the SP (p<0.01), were genotyped (Illumina Veracode platform) in a second independent replication population (RP) – these 380 healthy bank employees had undergone repeated 24-hour ambulatory BP monitoring. Association analyses were performed using additive genetic models. Quantile-quantile plots showed enrichment for significant P-values in both populations. The top hits are in the SLC17A8 and the GABR1 genes. SLC17A8 encodes a sodium-dependent inorganic phosphate cotransporter and GABRR1 encodes the subunit of the ionotrophic gamma-aminobutyric acid receptors. These genes have not previously been implicated in the causation of hypertension though the influence of the ANS on BP makes the observed associations biologically very plausible.

Ulster Med J. 2010 Sep;79(3):144.

P26. Mutations in the Human Zinc Finger Transcription Factor 8 (TCF8) Gene in Keratoconus and Corneal Endothelial Dystrophies Support a Genotype-Phenotype Correlation

D Muszynska 1, DP Dash 1, J Lechner 1, D O'Prey 1, D Frazer 2, J Moore 3, G Silvestri 2, J Jackson 2, AE Hughes 4, CE Willoughby 1,2

Purpose: Mutations in the human zinc finger transcription factor 8 (TCF8) gene have been reported in posterior polymorphous corneal dystrophy (PPCD) and Fuch's endothelial dystrophy (FECD). Although PPCD and keratoconus (KTCN) involve different layers of the eye, PPCD has been associated with KTCN in several reports. To investigate the role of TCF8 mutations in the pathogenesis of keratoconus, mutational analysis was performed in 70 unrelated individuals with KTCN.

Methods: The coding regions of TCF8 were PCR amplified, Sanger sequenced and analysed using Sequencher 4.7. Novel variants were screened in 100 unrelated population controls (200 chromosomes).

Results: A novel, heterozygous, missense mutation in TCF8 was identified in a patient with familial KTCN and absent from 200 control chromosomes. The mutation, c. 1920G>T, results in a non-conservative substitution of a highly conserved glutamine to histidine (p.Q640H). The mutation was detected in two siblings with KTCN and in their mother: a patient with FECD. The glutamine (G640) is an invariant residue in the homeodomain ofTCF8.

Conclusions: This data supports a strong genotype-phenotype correlation within the TCF8 mutational spectrum. Missense mutations in TCF8 result in keratoconus and FECD while nonsense and frameshift mutations result in PPCD.

Ulster Med J. 2010 Sep;79(3):144.

P27. Evaluation of eight candidate genes located within chromosome 6q22-q27 for association with glomerulonephritis in a UK population

DR Vanc 1, AP Maxwel 1, AJ McKnigh 1

There is an inherited predisposition to glomerulonephritis (GN) which is a leading cause of end-stage renal disease. We examined eight biological and positional candidate genes located at 6q22-27, including GJA1, IGF2R and the RAET1 family, for association with GN.

DNA was obtained from the national MRC-KRUK bank for GN (nmax=2,964) and genotyped using a case-control approach. Initially, individuals with GN (cases, n=583) were compared to individuals with no evidence of renal disease (controls, n=508). Potentially functional SNPs were identified from Ensembl. SNP genotype was downloaded from the International HapMap Project and tag SNPs selected using Haploview where SNPs were in Hardy-Weinberg equilibrium, r2>0.8 and minor allele frequency >0.05. SNPs (n=53) were genotyped using TaqMan and MassARRAY® iPLEX Gold technology. Data were analysed in PLINK using the test for trend.

Investigating all common and potentially functional variants revealed statistically significant association for rs9397449 and rs9397070 in RAETIG (P=0.001 and P=0.04 respectively). RAET 1G is an MHC class-1 related gene (6q24.2-q25.3) and SNPs in this region have recently been associated with diabetic nephropathy. Association was also suggested for IGFR2 at rs3734181 (P=0.01).

This data suggests that genes within 6q22-27 influence glomerulonephritis and we are presently conducting independent replication studies to validate our findings.

Ulster Med J. 2010 Sep;79(3):144.

P28. Identification of a novel locus for autosomal dominant Restless Legs Syndrome

EB Skehan 1, MMA Abdulrahim 1, NA Parfrey 1, CK Hand 1

Introduction: Restless Legs Syndrome (RLS) is a common neurological sleep-related disorder, affecting up to 15% of the general population. The disorder is characterised by an irresistible urge to move the lower limbs, accompanied by uncomfortable sensations, occurrence at rest, improvement with activity and worsening of the symptoms in the evening or at night. Linkage analysis has identified six genomic regions. No causative gene has been identified. Association mapping has highlighted a further five areas of interest.

Aim: To map and identify the gene responsible for RLS in an Irish family (RLS3002).

Method: Eighteen members of the RLS3002 family participated in the study; eleven affected and seven unaffected members. All known RLS loci and associated regions were examined for linkage. A genome wide linkage analysis scan was conducted.

Result: Linkage was excluded from published loci. The genome-wide scan identified a region of linkage with a maximum LOD score of 3.59, ( =0.00). A genetic region of 2.5 Mb was defined by haplotype analysis. Candidate genes have been identified and are the subject of further study.

Conclusion: We have successfully identified a novel locus for Restless Legs Syndrome, which will enable us to attempt to identify the causative gene in this family.

Ulster Med J. 2010 Sep;79(3):144.

P29. Implantation and Pregnancy Outcome In Relation to Inherited Coagulopathties

R Habibi 1, M Shiva 2, P Mokhtari 1, H Gourabi 1

Thrombosis interfere with feto-maternal interaction in the site of fetus implantation, progression of fetal growth and pregnancy termination. These situations are seen in relation to spontaneous pregnancy or artificial reproductive technology (ART). .Many inherited mutations and polymorphisms are attributed to this status. Some of these mutations are seen in factor V Leiden, prothrombin, and methylenetetrahydrofolate reductase. We selected 48 women with pregnancy complications included recurrent abortion, IUFD, and ART failure. Among 13 women were investigated for factor Leiden, 5 patients were positive (38.46%), 75% were positive for MTHFR (12/16), four cases are homozygote for these gene, and 8 cases are heterozygote. None of the 19 women tested for prothrombin was positive for mutation of this gene.

Although our samples are very small, our results are in Hardy-Weinberg equilibrium for MTHFR gene mutation. This study is continued with large population and in comparison with control group to confirm effects of these mutations in implantation and pregnancy outcome in our population.

Ulster Med J. 2010 Sep;79(3):144.

P30. Identification of novel STRA6 variants in patients with anophthalmia-microphthalmia-coloboma

J Casey 1, J Conroy 1, R Regan 1, N Shah 1, SA Lynch 2, A Green 1,2, S Ennis 1,2

Anophthalmia (absence of an eye), and the related condition microphthalmia (small eye), are relatively rare disorders estimated at 3 and 14 in 100,000 births respectively. The aetiology of anophthalmia/microphthalmia (AM) is not well understood but observed familial clustering suggests a significant genetic component. Several genes have been implicated in AM but, to date, mutations in these genes account for less than 25% of cases.

We report studies of an extended Irish family from an endogamous nomadic group presenting with various forms of AM. We employed homozygosity mapping to search for causative mutations in the affected members of the pedigree. Previous analysis excluded any causative mutation in known AM genes. Genotyping was undertaken using the high density Illumina 1M SNP platform. Homozygosity mapping identified a 0.9Mb homozygous segment on chromosome 15q22.32 shared by all 6 affected individuals but not shared by unaffected relatives. The candidate region was isolated by NimbleGen's target enrichment service and sequenced on the Illumina Genome Analyser.

We identified 3 homozygous variants (2 non-synonymous and 1 utr) in STRA6. a membrane protein involved in the metabolism of retinol. One of the NS variants is a double nucleotide polymorphism (DNP), a newly recognized source of genetic variation that is predicted to play a major role in disease predisposition.

Ulster Med J. 2010 Sep;79(3):145.

P31. Targeted Sequence Capture and Next Generation Sequencing of a 5Mb Region on Chromosome 15q Previously Linked to Keratoconus

J Lechner 1,A, DP Dash 1,A, G Silvestri 1,A,2, J Jackson 1,A,2, DG Frazer 2, AE Hughes 1,B, DAC Simpson 1,A, CE Willoughby 1,A,2

Purpose: Our group previously mapped a large Northern Irish family affected by autosomal dominant, clinically severe keratoconus and anterior polar cataract to a 5.5Mb region on chromosome 15q22. The linkage region contains 85 candidate genes and in 28 no pathogenic mutation was identified by Sanger sequencing. To identify the molecular genetic defect in this family targeted sequence capture and next generation sequencing (NGS) was performed.

Methods: A custom Nimblegen sequence capture array was designed to capture 5Mb of the 5.5Mb region (a 0.5Mb repetitive region was excluded). NGS of the enriched region was performed (Genome Analyzer) and reads aligned to a reference sequence using ‘Genomics Workbench’ software (CLC bio).

Results: Twenty-five potentially-pathogenic coding sequence variants were identified in affected family members. These variants were present in eleven known and predicted genes within the linkage region. Variants in seven genes were excluded following Sanger sequencing on the basis of non-segregation, presence in population controls and failure to replicate NGS data. Analysis of the remaining four genes is ongoing, in addition to intronic variants surrounding splice sites.

Conclusion: Custom targeted sequence capture followed by NGS of a linkage region is an effective strategy for disease gene discovery.

Ulster Med J. 2010 Sep;79(3):145.

P32. ADAR RNA editing in neurodegenerative diseases

Simona Paro 1,2, Leeanne McGurk 1, Aruna Raja 1,2, Hui Sun 1,2, Brendon Noble 3, Paul Heath 4, Paul Ince 4, Pamela Shaw 4, James Brindle 1, Mary O'Connell 1, Liam Keegan 1,2

In humans ADAR RNA editing controls key properties of excitatory glutamate receptors. ADARs convert specific adenosines to inosines in transcripts and inosine is read as guanosine during translation. Loss of RNA editing is implicated in neurodegeneration and glutamate excitotoxic neuron death in stroke and ALS. I will present our work on RNA editing loss in ALS motor neurons and on human mutations in ADAR proteins.

Drosophila also provides an excellent study model; in Drosophila ADAR RNA edits more than fifty known neuronal transcripts. Adar mutants are locomotion-defective with age-dependent vacuolisation in brain and retina. There is no extensive neuronal apoptosis. Instead intracellular membrane structures resembling those seen in autophagy mutants and in human lysosomal storage diseases appear and large fluid-filled vacuoles develop. The reduced viability at eclosion in an Adar null mutant is rescued by heterozygous Tor mutants which lead to increased autophagy. Overexpression of Atg5 also rescues the reduced viability as well as the locomotion defects, neurodegeneration and the reduced longevity of the Adar null.

Increased autophagy is clearly protective in the Drosophila Adar neurodegeneration and possibly also in human neurodegenerations associated with loss of ADAR RNA editing if underlying molecular mechanisms are conserved.

Ulster Med J. 2010 Sep;79(3):145.

P33. The impact of the MTHFR 677>T polymorphism on RUNX1 DNA methylation patterns

N Carroll 1, A Parle-McDermott 1

Folate/riboflavin status in combination with the MTHFR 677C>T genotype have previously been identified as important factors for consideration in health and disease. We identified RUNX1 as a responder to changes in folate/riboflavin status and considered DNA methylation as the mediator of this response. In the present study we assessed whether DNA methylation in the proximal promoter of RUNX1 correlated with MTHFR 677C>T genotype. DNA methylation within a CpG island of the proximal promoter of RUNX 1 was assessed by Methylation-sensitive high resolution melting (MS-HRM) in a panel of DNA samples from the Coriell lymphoblast collection. Comparison of the DNA methylation profiles of each genotype group shows that the CC and CT groups have a broadly similar pattern. The TT group, however, shows a dramatic enrichment of samples with 0% DNA methylation of their RUNX1 proximal promoter. The methylation profile of the TT group was compared to a combined CC/CT profile by Mann-Whitney test using SPSS yielding a P-value of 0.06 i.e, not significant. In conclusion, TT individuals may tend to exhibit 0% DNA methylation of their proximal RUNX1 promoter compared to CC or CT individuals particularly in the context of nutritional status. However, this requires further investigation.

Ulster Med J. 2010 Sep;79(3):145.

P34. No correlation between unmetabolised folic acid levels and folate gene polymorphisms in an Irish population

A MacCooey 1, MR Sweeney 2,3, A Boilson 2,3, S Minguzzi 1, JM Scott 4, A Staines 2,3, C Kelleher 2, L Daly 2, SW Bailey 5, PB Alverson 5, JE Ayling 5, A Parle-McDermott 1

Folic acid is an essential nutrient required for growth and development with many health benefits, particularly the prevention of neural tube defects. This has led to liberal voluntary fortification of a wide range of foods within Ireland. The increase in folic acid consumption has resulted in the appearance of circulating unmetabolised folic acid in the blood stream of the majority of the population. The risks and/or benefits associated with this are unclear. We wished to investigate whether circulating unmetabolised folic acid levels are influenced by genetic factors. We examined three functional and/or disease associated polymorphisms within genes involved in folate metabolism. These included the MTHFR (methylenetetrahydrofolate reductase) 677C>T, the DHFR (dihydrofolate reductase) intron A 19bp deletion/insertion polymorphism (DIP) and another DIP within intron 7 of MTHFD1L (mitochondrial 10-formyltetrahydrolate synthetase) c.781-6823ATT[7-9]. The polymorphisms were genotyped in 138 individuals from an elderly Irish cohort (aged 60-86 years recruited from the Lifeways study, a longitudinal study commissioned by the Health Research Board in 1999). Although trends were apparent, statistical analysis indicated that there was no significant correlation between polymorphism genotypes and unmetabolised folic acid. Further investigation is required in order to reach a definitive conclusion.

Articles from The Ulster Medical Journal are provided here courtesy of Ulster Medical Society

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