news

Inside this issue

ARTICLES• Coeliac Gene Testing

• An Introduction to Next-Generation Sequencing

• Pathology North Updates

Incorporating:• Northern NSW• Hunter New England• Mid North Coast• Central Coast• Northern Sydney

WINTER 2014

There are so many exciting events occurring across Pathology North; both within the organisation and directed onto it; that it is difficult to decide where to start this editorial.

The first comments should probably reference the Commonwealth Budget

impact upon public pathology services. It would, of course, be

advantageous to all if we knew what these would be, but without clarity and ratification of the budget items, Pathology North can only speculate, along with many others, about the impacts. Like all health providers we are bracing for a bumpy ride.

Specific issues will not be clear for some time however and we

expect no adverse impacts upon out strategies for enhancing our

services.

Our commitment to enhanced services is evident in our capital contribution to equipment and facility refurbishment across our client Local Health Districts. Most of these endeavours have been performed with generous assistance from the Local Health District and the Ministry of Health.

The Lismore Hospital laboratory facility was officially opened in April and is an exciting initial development in the upgrading of clinical facilities for Lismore Base Hospital. Grafton Hospital has a new laboratory facility now being built with occupation by the laboratory planned for July 2014. The Tweed laboratory has recently finalised a refurbishment of facilities with establishment of a new anatomical pathology component on site. Kempsey Hospital redevelopment is in the late stages of planning with a new pathology laboratory an important element of this project. Coffs Harbour laboratory is built and staff are settling into the new space adapting their equipment and services to fit.

Central Coast hospitals have been included in NSW Government plans for enhancement and building on the Gosford Hospital site for an extra approximately 100 beds will commence later this year. As part of this program new laboratory facilities will be developed on site.

Hornsby Hospital development phase 1 program is now proceeding and Pathology North will occupy new laboratories within this project at an early stage. This early engagement will ensure continuity of service for the Hornsby Hospital over the future development and create certainty of planning and alignment for the next phases of development here with the pathology laboratory established early and central to Emergency Department, ICU and operating theatre environments.

Along with these exciting capital developments Pathology North has moved further to harmonise testing platforms in chemistry, immunology, serology, transfusion/cross-match and coagulation services with all laboratories moving into onto common systems.

A single, common quality system is being implemented across all sites and IT evolution is occurring across our laboratories to unify our services even closer. Indeed the Central Coast laboratories have successfully transitioned onto our AUSLAB Laboratory Information System (LIS) over the later part of 2013 and movement for universal electronic request ordering across all AUSLAB sites is progressing well.

Changes to laboratory service times, to accommodate clinical service requests, are also in stages of development and evolution across many of our regional laboratories and negotiation with our staff about these requests are proceeding enthusiastically.

All of these programs are designed to enhance our service and to reinforce our commitment to clinical service within and across our client environments, be they community or Local Health District. We thank all of our clients and patients for their support and wish them well. Pathology North via any mechanism is always happy to hear any ways by which it may improve its contribution to the health of patients across New South Wales.

Dr Stephen G Braye Network Director Pathology North

FROM THE DIRECTOR

2Front Cover: Jared Lane, Scientist Molecular Genetics Royal North Shore Hospital

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Dr David Tran, BMedSci, MBBS

Clinical immunology and immunopathology registrar Pathology North, John Hunter Hospital

Dr David Tran graduated from Monash University with previous training in Melbourne and Brisbane. He has an interest in anaesthetic drug testing, autoimmune diseases and patient symptom score assessments.

A/Prof Glenn Reeves MB BS (Syd) FRACP FRCPA M Med Stat

A/Prof Glenn Reeves is a senior Staff Specialist in Immunology and Immunopathology working in public and private clinical and laboratory practice (Hunter Health, Pathology North, Coastal Immunology), with a particular interest in autoimmunity, both systemic (e.g. lupus) and organ-specific (including coeliac disease).

A/Prof Glenn Reeves is also Director of the Autoimmune Resource and Research Centre, a patient-focused charitable organisation offering information, support and research related to autoimmune conditions.

Coeliac Gene Testing Dr Glenn Reeves and Dr David Tran

Key Points:

• Coeliac HLA gene testing is not diagnostic for coeliac, as the permissive alleles are seen in > 20% of the population

• Negative coeliac HLA gene testing substantially lowers the likelihood of coeliac being present

• Coeliac HLA gene testing has particular utility in settings where endoscopic confirmation of coeliac is not desirable (e.g. paediatric populations, anticoagulant therapy) or in situations where patients are averse to discontinuing an already-established gluten-free diet to clarify duodenal biopsy appearances

Coeliac disease (CD) is a common (prevalence ~ 1%) immune condition where the bowel, sometimes along with other organs, is damaged by the body’s response to gliadin (gluten) contained in wheat, barley, and rye. In order to develop clinical features such as malabsorption (diarrhoea, steatorrhoea, nutrient deficiencies [e.g. vitamin D, iron]), patients with coeliac disease typically have a specific HLA genotype. Human leukocyte antigens (HLA) are the human component of the Major Histocompatibility Complex Molecule (MHC) which play an essential role in displaying peptides (self or foreign) to antigen specific T lymphocytes. This HLA-DQ2/DQ8 genotype facilitates presentation of gliadin-derived antigens to T cells, which then recruit other immune cells to mediate the bowel damage seen in coeliac sufferers, as well as serological changes (including formation of antibodies to tissue transglutaminase (TTG) and deamidated gliadin peptide (DGP)). Proving the presence of coeliac disease requires the demonstration of characteristic changes in the villous lining of the small bowel in individuals ingesting gluten – diagnosis is ideally reliant upon these histological features, with clinical features, serological changes, and genotype providing supportive, surrogate information.

It is generally acknowledged that coeliac disease rarely develops without the presence of a predisposing (“permissive”) HLA Class II DQ genotype. The HLA-DQ molecule is a heterodimer containing an alpha-chain (encoded by DQA1) and a beta-chain (encoded by DQB1). HLA nomenclature is evolving away from serological typing (e.g. DQ8) towards detailed molecular typing (DQA1*0301/DQB1*0302) that reflects the polymorphic sequence variation within the HLA genome. This change in nomenclature can be found in the medical literature and progressively in diagnostic pathology reports.

Further resolution of the permissive genes allows typing for such alleles as HLA-DQ2.5 (DQA1*0501/ DQB1*0201) and HLA-DQ2.2 (DQA1*0201/ DQB1*0202). Currently, the following HLA genotypes are associated with a genetic

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potential to develop coeliac: HLA-DQ2.5, HLA-DQ8, and HLA-DQ2.2 (see Table 1) 1. HLA-DQ2.2 has been added to this list of permissive alleles relatively recently, although it appears that the increase in risk conferred by this allele is not as great as that for the “classic” alleles (DQ2.5, DQ8)2. Given the negative predictive importance of CD HLA genotyping, these more recently established HLA associations have been added to the report, as relevant.

Table 1: Permissive alleles in coeliac disease

Permissive Alleles DQA1 DQB1 Risk3

DQ 2.5 cis DQA1*0501 DQB1*0201 PermissiveDQ 2.5 trans DQA1*0505 DQB1*0202 PermissiveDQ8 DQA1*03xx DQB1*0302 Permissive

DQ2.2 DQA1*0201 DQB1*0202 Somewhat Permissive

The primary role of CD genotyping in suspected coeliac is to provide high negative predictive value. In other words, if none of the permissive CD HLA genotypes are identified in the context of possible CD, then the likelihood of this diagnosis drops substantially, to a level around 0.5%, hence prompting the need to redirect diagnostic focus. A negative CD HLA genotype (i.e. none of the permissive alleles identified) is useful in substantially reducing the likelihood of coeliac being an explanation for the patient’s symptoms.

In order to simplify interpretation of coeliac gene reports, Pathology North: Hunter Immunology provides a summary statement indicating whether the detected genotype includes potentially permissive HLA alleles for coeliac. For those interested in the specifics of the genotype, a detailed summary of the HLA-DQ typing is available upon request.

A positive CD HLA genotype means that it is possible for the patient to develop coeliac disease, but the result is not inherently diagnostic in itself. To diagnose CD requires appropriate gluten-responsive clinical features in the context of suggestive serology and biopsy findings.

References:

1) Mubarak A, Spierings E, Wolters V, van Hoogstraten I, Kneepkens CM, Houwen R. “Human leukocyte antigen DQ2.2 and celiac disease”. J Pediatr Gastroenterol Nutr. 2013 Apr;56(4):428-30

2) Sollid LM, Qiao SW, Anderson RP, Gianfrani C, Koning F. “Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules”. Immunogenetics. 2012 Jun;64(6):455-60.

3) Megiorni F, Mora B, Bonamico M, Barbato M, Nenna R, Maiella G, Lulli P, Mazzilli MC. “HLA-DQ and risk gradient for celiac disease”. Hum Immunol. 2009 Jan;70(1):55-9

Testing aspects

Coeliac Gene Testing is performed on whole blood (EDTA, ACD). Heparin collection tubes are not accepted. Please store and transport at room temperature. Assay performed weekly. Medicare rebate available for the test (71151)

Breanna L. Peacock operating the “Luminex” patented technology.

Contacts for further information

Pathology North Hunter (Immunology)

Pathologist on call: 0423 843 929

Laboratory: 4921 4018

Director: Dr Theo deMalmanche

Pathologists: Dr Glenn Reeves, Dr Kathryn Patchett

Registrar: Dr David Tran

Dr Lan Nguyen, BMedSci(Hons), MBBS, MAACB

Chemical Pathology Registrar

Dr Nguyen has recently joined Pathology North. She has previously trained at St Vincent’s Hospital, The Children’s Hospital at Westmead,

Douglass Hanly Moir and Bolzano Central Hospital Laboratory (Italy).

Professor Leslie Burnett, MBBS, MSc(Bioinf), PhD, DBA, FRCPA, MAACB, FHGSA, FFSc(RCPA)

Biochemistry and Genetics

Professor Burnett is a clinical pathologist, specialising in Chemical

Pathology/Clinical Chemistry, Genetic Pathology and Bioinformatics. Professor Burnett holds a number of senior professional roles both in Australia and internationally. He is currently a member of the Human Genetics Advisory Committee of the National Health and Medical Research Council (NHMRC) of Australia (by Ministerial appointment) and Chairman of Lab Tests Online Australasia. His past roles have included serving as Chairman of the National Pathology Accreditation Advisory Council (NPAAC) of Australia, President of the Australasian Association of Clinical Biochemists (AACB), Chairman of the Quality Assurance, Scientific and Education Committee (QASEC) and Councillor of the Royal College of Pathologists of Australasia (RCPA). Dr Burnett is Clinical Professor in Pathology and Genetic Medicine within the Northern Clinical School of the Faculty of Medicine, and Honorary Associate in Bioinformatics within the School of Information Technologies, University of Sydney. Professor Burnett was the Director of PaLMS from 1997 to 2007 and Chief Executive of Pathology North from 2007 to 2010.

An Introduction to Next-Generation SequencingBy Dr Lan Nguyen and Professor Leslie Burnett

The “genomic era” was heralded in 2003 with the completion of the Human Genome Project. It took more than 13 years to sequence the 2.85 billion nucleotide human genome at a cost of US$2.7 billion. Yet today, just 11 years after this landmark achievement, it may be possible to sequence over 18,000 human genomes a year at a rate of more than one genome an hour, and at the long-sought-after cost of $1,000 per genome. This dramatic reduction in both costs and analysis time has been enabled by new sequencing technology called next-generation sequencing (NGS) or massively parallel sequencing (MPS).

Until now, DNA sequencing has been done by traditional “Sanger sequencing” or 1st generation sequencing. This involves sequencing of a DNA template using dye-labelled terminator nucleotides, followed by separation and detection of each individual sequencing reaction. In contrast, NGS involves sequencing of hundreds of thousands to millions of DNA templates simultaneously, or ‘in parallel’, with the sequencing reaction coupled with detection. This has resulted in going from an output in the order of kilobases (103) per run up to giga- (109) and more recently, terabases (1012) per run.

Though the various NGS platforms from different manufacturers use differing technologies, the general steps involved are similar:

1. Preparation of a sequencing library

2. Immobilisation of the sequencing library to a solid surface

3. Amplification (not required for some technologies)

4. Sequencing reaction and signal detection

5. Data analysis

At the Pathology North Royal North Shore Hospital campus, we are using the Ion Torrent (Life Technologies) platform. This NGS platform performs its DNA sequencing by detecting on an electronic chip tiny changes in pH produced by the DNA sequencing reactions (Figure 1). Briefly, DNA is fragmented and adaptors are ligated to the ends, creating the ‘sequencing library’. This DNA library is mixed with microscopic ‘capture beads’, polymerase

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chain reaction (PCR) reagents and an emulsion oil to create water droplets, with each droplet containing a single bead and a single library fragment which undergoes PCR (‘emulsion PCR’). Beads now coated with millions of copies of DNA are put onto an electronic chip containing up to 7 million microscopic wells, each well-being only just large enough to be able to accommodate a single coated bead. In each sequencing cycle, reagent containing one of the four bases (A, T, G or C) is washed over the chip and if the base is incorporated into the DNA template, there is release of a proton (H+). The resultant change in pH (~0.02 pH units per base incorporation) is detected in each well and is converted to a digital signal, which is electronically interpreted as a ‘base call’, from which the DNA sequence of the template can be deduced. In successive analytical reaction cycles, each lasting only a few minutes, the different nucleotides are washed over the chip in a defined order to produce a base sequence or ‘read’ for each bead.

NGS may be used to sequence different nucleic acid targets:

• Whole genomes e.g. to identify somatic mutations in cancers to aid in cancer diagnosis, prognosis or treatment

• Whole exomes e.g. to identify mutations causing Mendelian disorders

• Transcriptomes (the RNA sequence including mRNA and tRNA) e.g. to identify overexpressed genes in cancers

• Mitochondrial genomes e.g. to identify mutations causing mitochondrial disorders.

In our laboratories at the Pathology North Royal North Shore Hospital campus, NGS is already being used for targeted gene sequencing. We are a national reference centre for diagnosing and reproductive/pre-natal screening in at-risk individuals for a number of potentially fatal, autosomal recessive inherited disorders, such as Tay-Sachs disease, Canavan disease, familial dysautonomia (Riley-Day syndrome), Bloom syndrome and Niemann Pick disease type A. Through our efforts, Tay-Sachs disease has been effectively eliminated from those communities in Australia who are ancestrally at greatest risk: there has not been even a single case of Tay-Sachs disease in Australia for the last 15 years in any family who undertook genetic testing for this incurable and fatal childhood condition. We also undertake statewide testing for many adult variants of the cystic fibrosis (CFTR) gene, particularly for the rarer variants not easily detected using conventional DNA sequencing. Our laboratory can often find significant

DNA variants that may be missed or overlooked by less-specialised testing services.

It has been remarked that though the raw chemical costs to sequence a genome may be “only” $1000, its subsequent analysis and interpretation may cost $10 000 or even $100 000! The average person will differ from the reference human genome at a rate of one variant per 103 nucleotides, with most of these variants being of no clinical significance; noting that the genome is approximately 109 bases long, this means the average person will have one million variants, of which perhaps only a dozen may be relevant.

Powerful computational tools and sophisticated bioinformatics algorithms are required to condense the huge amount of terabyte-size data obtained from NGS to find and extract the clinically important information. Firstly, the millions of short DNA ‘reads’ must be reassembled back to form a continuous DNA sequence. This involves aligning the reads against a reference human genome sequence. Differences between the alignment and reference sequence are identified (‘variant calling’), filtered and annotated to identify those variants that may be clinically significant. False positive and negative variant calls may be made due to errors introduced during amplification or sequencing, from improper alignment or due to the algorithms used. Correct annotation is highly dependent on the accuracy of information obtained from databases, such as those containing known disease-causing mutations or common polymorphisms. Functional characterisation of novel variants is also difficult. The pathogenicity of a variant may be inferred by examining conservation across species or predicted changes in protein structure and function, but often the significance remains uncertain.

Finally, sequencing of whole genomes or exomes is unlike most diagnostic tests where the test performed is specific to the clinical indication. It is inevitable that there will be ‘incidental findings’, i.e. pathogenic or likely pathogenic alterations in genes that are unrelated to the clinical indication. The handling of incidental findings by laboratories is a topic of current intense international debate by both pathologists and ethicists. The emerging consensus seems to be that patients should have the right to opt out of being told the results of these incidental findings, but if they are to be reported then comprehensive pre- and post-test counselling needs to be given.

Despite these challenges, NGS is revolutionising genetic testing and research, and continues to evolve. The current NGS technologies and its related bioinformatics are constantly being improved upon and new technologies are

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on the horizon. For example, the MinION system (Oxford Nanopore Technologies) is a disposable, flash-drive sized USB device that can be plugged into a laptop computer, works directly with blood or serum and sequences single molecules using nanopore technology. There are also a growing number of new applications for NGS, such as whole genome newborn screening, which is undergoing pilot study in the USA. As there is wider adoption of NGS technologies in Australia, we will be seeing a dramatic change in the utilisation and clinical impact of genetic testing.

Work is now underway in our laboratory to develop a range of new tests for many conditions that, until now, have lacked clinically useful diagnostic tests. Working with our clinical colleagues, we are exploring tests for inherited cardiac and muscle disorders, as well as for mutations present in some brain tumours.

Further reading:

1. Lew RM, Proos AL, Burnett L, et al. Tay Sachs disease in Australia: reduced disease incidence despite stable carrier frequency in Australian Jews. MJA. 2012;197(11);652-654

2. Life Technologies 2014, www.lifetechnologies.com/au/en/home/brands/ion-torrent.html

3. Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem. 2013;6:287-303.

4. Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med. 2013;369(16):1502-1511.

5. Oxford Nanopore Technologies 2014, https://www.nanoporetech.com/technology/the-minion-device-a-miniaturised-sensing-system/the-minion-device-a-miniaturised-sensing-system

Figure 1. Steps in next-generation sequencing using Ion Torrent technology (PCR, polymerase chain reaction)

Genetics Laboratory Royal North Shore Hospital

Dr Lisa Koe, Anné Proos, Professor Leslie Burnett and Dr Lan Nguyen

Pathology North UpdatesLismore LaboratoryThe Pathology North, Lismore Laboratory relocated in April 2014, providing an opportunity to create a state-of-the-art facility at Lismore Base Hospital to support the hospital and the local community.

Note: Thank you to Northern NSW LHD Northern Exposure May 2014 for the photos.

Raymond Terrace Collection CentrePathology North, Hunter New England has opened a new collection centre in Raymond Terrace. The new collection centre is located in the new HealthOne GP Superclinic on 4 Jacaranda Ave and will be open Monday to Friday 8am - 4.30pm and 8am - 12pm on Saturday’s.

A trained paediatric and AS:4308 accredited urine drug screen collector will be available onsite. ECG and Holter services will be available by appointment.

The new Collection Centre will provide a well-equipped, spacious and easily accessible facility, with a comfortable, modern waiting area. A large car park is adjacent to the Super Clinic. Bulk-billing for Medicare eligible tests will be available, in line with the other Pathology North Collection Centres.

Next generation sequencing technology helping to screen for hereditary genetic conditionsPathology North, Royal North Shore Hospital, officially commissioned its latest next generation sequencing analyser to assist with testing and screening for genetic conditions.

The analyser is also part of an ongoing partnership with Wolper Jewish Hospital in Sydney.

Dr Stephen Braye, Network Director Pathology North shows guests around the Pathology Laboratory.

Professor Leslie Burnett and Dr David Golovsky (President and Chairman of the Board Wolper Jewish Hospital) at the Ribbon Cutting Commissioning Event

John Tranter, Operations Manager Northern NSW Pathology North with Tracey McCosker,

Chief Executive NSW and Fiona Robinson