Poster presentations: Molecular Genetics.

3.01

Localisation and fine mapping of a recombination hotspot on chromosome 19

Joanne Jones (1), A Cox (2), AG Wilson (1)

(1) Division of Genomic Medicine, University of Sheffield, Sheffield, (2) Institute for Cancer Studies, University of Sheffield Medical School, Sheffield

The rate of recombination across the human genome, calculated by comparisons between genetic and physical map distances, has been shown to be highly variable. Recent reports indicate the presence of recombination "hotspots", where the rate of recombination is significantly higher than that seen in other chromosomal regions. One indication of the presence of such a hotspot is the consequent breakdown of linkage disequilibrium in the region.

Recent analysis of chromosome 19p13 revealed patterns of linkage disequilibrium indicative of the presence of a recombination hotspot in a 1Kb region, defined by markers D19S406 and D19S427. Further data shows a disease-locus defining recombination event within this region. Recombination analysis of 19p13 was performed by genotyping both DNA from families and single sperm. Familial analysis is dependent on the availability of samples from both parents and at least 2 children. It is also dependent on the parents being heterozygous for the markers tested in order for inherited haplotypes to be determined. Single sperm typing is a powerful tool that eliminates the need for the collection of extensive family samples, although heterozygosity at the marker of interest is still essential. Initial genotyping data of 14 informative families has identified 1 recombination in 28 meioses. This is approximately equal to the expected recombination rate for a region of this size. Further analysis of 100 families is currentl y underway in order to increase the power of the study and prove the presence of a hotspot by identification of an elevated recombination rate. The refinement of the region of recombination, including the use of SNP mapping, will also be described.

joanne.jones@sheffield.ac.uk

3.02

Detection of point mutations in the Dystrophin Dp71 transcript using dHPLC.

Pierre Foskett, M Boxer, A Wilcox, D Wilcox

Medical Genetics, Yorkhill NHS Trust, Glasgow

Duchenne muscular dystrophy is a consequence of mutation in the dystrophin gene at Xp21.1. The gene is exceptionally large and fragmented with a total of 79 exons coding for a 12kb mRNA transcript. Around three-quarters of mutations in the dystrophin gene can be explained by gross deletion and duplication and are known to cluster around specific exons. The remainder are point mutations which are thought to occur randomly throughout the coding sequence. As a consequence, screening for these mutations is likely to be a task of considerable workload. We have applied automated dHPLC analysis to PCR fragments from the dystrophin gene allowing an increase in analytical throughput. Furthermore, it has been reported that there is an increase in the rate of mutation within the brain specific Dp71 transcript of dystrophin amongst individuals with mental retardation. Dp71 is coded by exons 63-79 of the dystrophin gene; reducing the amount of sequence to be screened. We used the criteria of a clinical description of ment al retardation associated with the muscular dystrophy to select individuals for this study. The results show that dHPLC is an effective screening method for the dystrophin gene. However the use of a basic definition of mental retardation failed to increase the level of mutation detected within this portion of the dystrophin gene.

molgen@yorkhill.scot.nhs.uk

3.03

Frequency of the deletion allele of the P gene among different phenotypic groups of oculocutaneous albinism in Southern Africa.

Patricia Lund (1), J Newton (2), N Garrison (2), Z Yi (2), M Brilliant (2)

(1) Cell and Molecular Biosciences, Coventy University, (2) Department of Pediatrics, University of Arizona Health Sciences Center, USA

Oculocutaneous albinism (OCA) is a relatively common genetic condition in Africa, with a frequency of 1 in 4000 among the Shona and Ndebele people of Zimbabwe and 1 in 1500 among the Sotho of northern South Africa, where traditional marriage customs encourage consanguineous unions. OCA results in hypopigmentation of the hair, eyes and skin. A 2.7 kb deletion of the P gene is associated with OCA2 in people of African ancestry. This study examined the presence of the deletion mutation in 34 subjects with OCA in northern South Africa using a PCR based assay. Of the 68 chromosomes, 43 (63.2%) showed the deletion, with 18 individuals homozygous and 7 heterozygous for this mutation. No deletion allele was detected in the remaining 9 cases. There are two common phenotypic subgroups, distinguished by the presence or total absence (the phenotype shown by all the homozygotes for the deletion allele) of pigmented patches or nevi on sun exposed parts of their skin. Two OCA subjects belonging to the Venda ethnic group wit h a rare phenotype (confluent freckles covering sun exposed skin) did not have the deletion allele. These may have another mutation of the P gene, or represent a different type of albinism. The high frequency of this one mutation allele supports the hypothesis that the deletion is of African origin, with a common founder, that has spread through central and southern Africa with the migration of the Bantu speaking people.

p.lund@coventry.ac.uk

3.04

Clinical and Genetic Spectrum of CADASIL in three Arab Families

Khaled Abu-Amero, S. Bohlega, A. Al-Shubili, M. Kambouris

King Faisal Specialist Hospital, Riyadh, Saudi Arabia

Introduction: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leucoencephalopathy (CADASIL) is an inherited arterial disease of the brain resulting in stroke. The disease has been described in Europe, North America and Far East, but not the Middle East. The defective gene in CADASIL is Notch3 gene that maps Hsa 19.

Methods: Notch3 exons (3, 4, 11, 12, 18 and 19) were screened for mutations by MDE heteroduplex analysis. Apparent heteroduplex patterns were sequenced for identification of the pattern causing sequence alterations.

Results: Three families (from Kuwait, Saudi Arabia, and Sudan) with 16 affected individuals were studied. Clinical presentations include migraine and repeated strokes around the age of 30, leading to severe disability and subcortical dementia at age 45 years. Four siblings were asymptomatic with MRI examination revealing white matter disease. Six affected individuals died at around the age of 50. Mutations screening of exons 3, 4, 11, 12, 18 and 19 in the Notch3 gene, where >90 % of the reported mutations exist, showed the presence of heterozygous 406 C<

Conclusion: This is the first description of CADASIL in Arab families with a clinical phenotype and genotype similar to other ethnic groups. The third family is currently being screened for less common Notch3 mutations.

kamero@kfshrc.edu.sa

3.05

Human ZFH-4, a Candidate Gene for Congenital Bilateral Isolated Ptosis

Rachel Keay (1), TFW McMullan (1), AD Tyers (2), JA Crolla (1), DO Robinson (1)

(1) Wessex Regional Genetics Laboratory, (2) Department of Ophthalmology, Salisbury District Hospital, Salisbury

Ptosis (blepharoptosis) is the abnormal drooping of the upper eyelid and can impair full visual acuity. Two linkage loci have been reported for congenital bilateral isolated ptosis, an autosomal dominant locus at 1p32p34.1 and an X-linked dominant locus at Xq24-Xq27.1.

An isolated ptosis patient was found to carry a de novo balanced translocation, 46, XY, t(1;8) (p34.3;q21.12). The chromosome 1 breakpoint does not disrupt a gene, however the chromosome 8 breakpoint interrupts a human homologue of the mouse zfh-4 gene, encoding a zinc finger homeodomain transcription factor. Human ZFH-4 consists of 10 exons spanning ~161kb of DNA. The translocation breakpoint occurs in intron 4 resulting in truncation of the gene and its protein product.

In mice, zfh-4 is expressed in neurones in the mid and hind-brain during embryogenesis. Therefore reduction in expression may affect the development of the oculomotor nuclei situated in the midbrain. The oculomotor nerves could be affected including the innervation of the levator palpebrae superioris (LPS), which is defective in ptosis in humans. To determine whether ZFH-4 is truly associated with ptosis, we are currently investigating a cohort of patients with congenital bilateral isolated ptosis in an attempt to identify additional ZFH-4 mutations.

wessex.genetics@dial.pipex.com

3.06

Mutation screening of the UBE3A gene

Nicola Craig, A Cooke, J Tolmie, M Boxer

Department of Medical Genetics, Yorkhill Hospital, Glasgow, Scotland

Angelman syndrome (AS) is associated with an imprinted region of chromosome 15q11-q13 and results from non-maternal expression/ inheritance of genes in this region and occurs at a frequency of 1/15,0001/20,000 births. Mutations in the UBE3A gene, encoding E6-AP ubiquitinprotein ligase, occur at a reported rate of 4-10% of AS patients. A screen for mutations within this gene was established for AS patients who had been demonstrated to display a normal inheritance pattern for this chromosomal region (i.e. no deletion, UPD or imprinting centre mutation). This screen involves PCR amplification of all 10 coding exons from this gene (including the splice junctions) followed by sequencing. To date 5 patients have been screened in this way and 3 mutations identified in 3 patients. The types of mutations identified include insertions and deletions. Patients heterozygous for a deletion or insertion, and for which the genetic change was not clear from sequencing alone, had their PCR product cloned and then sequenced. Th is allowed the isolation of each allele and therefore, clearer identification of the sequence change. Future work will include the establishment of WAVE-DHPLC pre-screen for this assay.

molgen@yorkhill.scot.nhs.uk

3.07

The role of SOD1 mutations in young-adult motor neuron disease

Pamela Renwick (1), Matthew J Parton (2), Nigel Leigh (2), Christopher Shaw (1, 2), Stephen Abbs (1)

(1) Genetics Centre, Guy's and St Thomas' Hospital NHS Trust, Guy's Hospital, London, (2) Dept of Neurology, Inst of Psychiatry, and Guy's, King's and St Thomas' School of Medicine and Dentistry, London

Motor neuron disease (MND) is a heterogeneous condition with individual cases varying in terms of age, site of onset, rate of progression and overall survival. Mutations in copper/zinc superoxide dismutase (SOD1) have been reported in 20% of familial and 2-3% of apparently sporadic cases. Familial cases and those with SOD1 mutations develop MND symptoms over 20 years earlier (mean onset age 45.7), than for MND overall. The indication for SOD1 mutation screening in young adult motor neuron disease patients (YAMND, aged 45 or under at first symptom) is however uncertain. Therefore, one hundred YAMND cases were collected prospectively and screened for SOD1 mutations by DHPLC analysis using a Transgenomic WAVETM analysis system. Twelve of fourteen YAMND families appear to exhibit autosomal dominant inheritance, though penetrance was incomplete in five. One family suggests autosomal recessive inheritance and another X-linked recessive u the latter is possibly relevant to the preponderance of sporadic male YAMND ca ses. SOD1 missense mutations were found in 3/14(21.4%) familial pedigrees and 2/86 (2.3%) in sporadic individuals, which agrees with previous surveys of patients presenting at any age. We conclude that SOD1 screening is not indicated in Y

Pamela.Renwick@gstt.sthames.nhs.uk

3.08

Mutation Screening for Autosomal Recessive Malignant Osteopetrosis

Alison Taylor (1), T Cranston (2), S Tabrah (1), G Norbury (1)

(1) Clinical Molecular Genetics, GOSH, (2) Molecular Genetics, Oxford

Autosomal recessive malignant osteopetrosis is a rare congenital disorder of bone metabolism affecting 1/200,000 individuals. Clinical features include fractures, visual impairment, nerve compression resulting in headaches, blindness and deafness, haematological difficulties, frequent infections and growth retardation. Osteopetrosis is generally diagnosed through skeletal X-rays with bones appearing unusually dense with a chalky white appearance. Mutations of the TC1RG1 gene (11q13) have been shown to cause ~50% of cases of autosomal recessive osteopetrosis. We are currently developing a diagnostic service for mutation screening of this gene in our laboratory. Direct sequencing of exons 2 to 20 of the TCIRG1 gene has been carried out on five affected patients. One patient was homozygous for c1031-2 ins G mutation in exon 9 and one patient was homozygous for the IVS13+2 T>A mutation. In two patients only one mutation has been identified: a 9bp deletion in exon 19 and a nonsense mutation (W304X) in exon 14. Pol ymorphisms were identified in nitrons 2 and 4, in the patient with the 9bp deletion. These acted as linked markers for prenatal diagnosis in this family. The identification of mutations in individuals affected with osteopetrosis will allow us to offer prenatal diagnosis by molecular analysis. At present the strategies currently available for prenatal diagnosis of osteopetrosis can only be attempted late in pregnancy and are subject to a high error rate.

tayloa@gosh.nhs.uk

3.09

PTT analysis of the dystrophin gene: a review of the mutation spectra

Shu Yau, Z Hatton, P Renwick, V Nihalani, S Abbs

DNA Laboratory, Genetics Centre, Guy's & St Thomas' Hospital NHS Trust, Guy's Hospital

Approximately two-thirds of DMD/BMD patients have a deletion or duplication of the dystrophin gene. Using fluorescent quantitative PCR analysis, we can detect approximately 98% of these mutations. To provide a molecular diagnostic service for the remaining one-third of patients with no detectable mutation we apply the Protein Truncation Test to screen the dystrophin coding sequence using muscle RNA. Dystrophin mutations were found in 30/33(91%) DMD patients, 3/10 (30%), BMD patients and 2/3 (67%) manifesting females. Gross out-of-frame rearrangements were detected in 3 DMD patients; an exon 18 duplication, an exon 61 deletion and a deletion of exons 64-74. In the remaining 30 DMD patients we detected 17 (57%) nonsense mutations, 6 (20%) insertions/deletions of one or more nucleotides and 4 (13%) splice site mutations, all of which are predicted to produce a truncated dystrophin protein. Three BMD patients had splice mutations that result in in-frame deletions, while 2 nonsense mutations were identified in man ifesting females. An analysis of the location of these 35 mutations suggested that there was no point mutation hotspot in the gene. Using the combined approach of quantitative PCR and PTT analysis, we have been able to detect approximately 97% of dystrophin mutations in DMD patients.

Normal PTT products were present in 3 DMD patients, 7 BMD patients and one manifesting female suggesting that they may have missense or promoter mutations. These patients are ideal candidates for the DNA based approach, which we are developing for the detection of all dystrophin mutations.

Michael.Yau@gstt.sthames.nhs.uk

3.10

A complex deletion-inversion-deletion event on Xp21 producing a hybrid IL1RAPL1/dystrophin gene product

Vandana Nihalani, JM Wheway, S Yau, D Ellis, M Irving, M Splitt, S Abbs, RG Roberts

DNA Laboratory, Genetics Centre, Guy's and St Thomas' NHS Trust, Guy's Hospital, London

In patients with contiguous gene syndromes the most common underlying pathogenic mutation is a simple deletion that disrupts the functions of two or more adjacent genes. However, in this study we describe a case where the mutation involves a complex rearrangement. The proband was diagnosed with a multifarious phenotype comprising Duchenne muscular dystrophy (DMD), glycerol kinase deficiency (GKD) and adrenal hypoplasia congenita (AHC) suggesting a large contiguous gene deletion on Xp21. While, cytogenetic analysis revealed a normal 46, XY karyotype, multiplex PCR deletion analysis identified two non-contiguous deletions. The first deletion involved the dystrophin gene where exon 52 only was deleted. The flanking exons 51, 53 and the rest of the 3'end of the gene were found to be present. The second deletion of 1.5Mb removed the GK and AHC genes, together with the MAGEB gene cluster, the testis-specific ferritin heavy chain gene, FTLH17 and part of the mental-retardation gene IL1RAPL1. Further characterization of the deletion breakpoints and RT-PCR analysis confirmed the presence of a complex rearrangement of Xp21 involving a deletion-inversion-deletion event. This results in a 600 kb inversion containing exons 53-79 of the dystrophin gene and formation of a hybrid IL1RAPL1/dystrophin gene that produces a chimeric IL1RAPL1/dystrophin transcript. This rearrangement was also found to be present in the mother of the proband and was grandpatemal in origin, suggesting that the mutation either arose as a result of inappropriate intramolecular recombination of the unpaired X chromosome in the grandfather or, early in embryogenesis in the mother.

Vandana.Nihalani@gstt.sthames.nhs.uk

3.11

Potential for false negative results in Spinal Muscular Atrophy carrier testing.

David Bourn (1), R Butler (2), D Cairns (1), F McGill (1), J Sibbring (1), R Mountford (1)

(1) Regional Molecular Genetics Lab, Liverpool Women's Hospital, (2) Institute of Medical Genetics, University Hospital of Wales, Cardiff

We are currently testing for Spinal Muscular Atrophy (SMA) carrier status using a fluorescent STS dosage assay to detect SMN1 deletions and partial gene conversions. It is not usually possible to completely exclude SMA carrier status (even without taking subtle SMN1 mutations into consideration) because a small but significant proportion of normal chromosomes carry two copies of SMN1. Published figures for the percentage of normal chromosomes with two copies of SMN1 are in the range of two to four percent. An individual with two copies of SMN1 may therefore still be a carrier of a deletion on one chromosome masked by the presence of two copies of SMN1 on the normal chromosome. We have now tested three families where obligate and potential carriers have been found to have two copies of SMN1 on one chromosome masking an SMN1 deletion. In addition we have been able to effectively exclude carrier status in potential carriers in another family using a combination of dosage and linkage analysis. These results show the usefulness of extended family studies in SMA carrier testing.

david.boum@lwh-tr.nwest.nhs.uk

3.12

Investigating the role of the APOC3 gene in hypertriglyceridaemia.

William Wright, IS Young, CA Graham, DP Nicholls

Centre for Cardiovascular and Genetic Research, Queen's University, Belfast

Hypertriglyceridaemia (HTG) is a common multi-factorial disorder with a major genetic component, affecting over 10% of the population, potentially increasing the risk of atherosclerosis. One of the candidate genes encodes apoClll, an inhibitor of lipoprotein lipase, a major protein involved in triglyceride metabolism.

Thirteen overlapping fragments covering ~6kb, spanning the exons, introns and regulatory regions of the APOC3 gene have been sequenced in 24 severely HTG patients (TG > 9 mmol/l) and 24 controls (TG < 2 mmol/l), with the aim to identify SNPs, which could affect the expression, splicing or coding of APOC3. A total of 37 SNPs were identified, of which 14 were novel, with 28 having a minor allele frequency > 5%.

Two of the SNPs, 3175C/G and 3206T/G, previously associated with HTG, were genotyped by RFLP (Sad and Bbvl respectively) in a control (TG < 2mmol/l, n=112) and a HTG (TG > …