New disc-based technologies for diagnostic and research applications

New disc-based technologies for diagnostic and researchapplications

Raj Barathur, Jack Bookout, Srinand Sreevatsan, John Gordon, Martina Werner,GautamThor and Mark Worthington

Burstein Technologies Inc., Irvine, California, USA

Correspondence to Raj Barathur, Burstein Technologies Inc., 163 Technology Drive, Irvine,California 92618, USA. E-mail: [email protected]

Received 22 January 2002; accepted 19 June 2002

The role of genotypic analysis in disease diagnostics and drug response assessment is continually expanding. New genomicdiscoveries combined with new, novel technologies may provide a greater range of testing capabilities in the near future. Wedescribe the application of nanotechnology, in which DNA microarrays have been placed in a microchannel environment thatcan be read and analyzed in an optical (CD/DVD) disc drive system. The potential exists to combine molecular andimmunological applications together into a rapid, low-cost, high-capacity screening platform. The relevance of this technologyis discussed in respect to infectious agent detection, pharmacogenomics, neurogenomics and genetic variations associated withneurologic diseases. Psychiatr Genet 12:193–206 r 2002 Lippincott Williams & Wilkins.

Keywords: CD/DVD discs, genotyping, gene variants, MEMS, microarrays, nanotechnology, neurogenomics, neurologicdisorders, optical discs, pathogen detection, pharmacogenomics

INTRODUCTION

The past decade has seen the rapid development ofBioMEMS (MicroElectroMechanical Systems) andnanotechnology, driven by ever-increasing screeningrequirements of the pharmaceutical/pharmaco-genomics industry and by long-held interests by themedical community for cost-effective point of carediagnostic instrumentation (Weber, 1997; Kleyn andWesell, 1998; Kost, 2000).

To meet these demands, many new plat-form technologies have been created, of whicha number are reviewed in this volume. The basisfor many of these advancements is the adaptation ofmicro-electronic technologies developed for the com-puting industry. For example, two major contribu-tions from this source are the microchipmanufacturing (from which MEMS fabricationtechnologies evolved) and optical informationand data storage devices (providing advances ininstrument miniaturization with increased analy-tical capacities) (Fodor et al., 1993; Kido et al.,2000).

Microchip and micro-array based platforms havereceived recent attention due to their relativelyhigh-capacity sequence interrogation capabilities,and a number of research groups and companies

have utilized this approach (e.g. Affymetrix Inc.,Genetic MicroSystems, Gene Logic Inc., IncytePharmaceuticals Inc.) (Bowtell, 1999). Relatively lessattention has been given to the parallel developmentof a smaller group of companies and researchersadapting optical disc technologies for biological andbiochemical analysis (Kiddo et al., 2000) (Gyros AB,Tecan-Gamera Bioscience, Abaxis Inc., BursteinTechnologies Inc.).

All of these groups have seen in compact disc(CD) technology the potential for developing rapid,low-cost, high-capacity screening systems byadapting existing industry standard technology. Bypackaging different assay formats on disc, the goalof achieving a generic format capable of suppor-ting multiple applications would be realized. Thepresent paper describes progress towards this endby Burstein Technologies Inc. (BTI) on molecularand immunological assays, and the future potentialof this technology. The platform described en-ables physicians/laboratories to use specially designedCD discs and a CD/DVD reader attached toan ordinary personal computer to deliver awide range of tests including clinical laboratorydiagnostics, biological warfare agent detection, for-ensic DNA tests, and food and water contaminationtests.

0955-8829 r 2002 Lippincott Williams & Wilkins Psychiatric Genetics 2002, Vol 12 No 4 193

Psychiatric Genetics 12:193–206

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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TECHNOLOGY

All of the BTI assays described are carried out withinan enclosed disc format read by a modified CD drive.The platform uses standard or semi-transmissive CDor DVD discs to deliver a wide range of tests using aCD reader attached to a personal computer. Thesystem will identify and quantify biochemical inter-actions inside a specially designed CD disc, theBioCompact Disc (BCD) (Table 1). A stepwisedepiction of the array utilization from hybridizedDNA at the molecular level to its detection in theBioCompact Disc reader (Figure 1) and a flowdiagram of the BCD drive function (Figure 2) areshown. The key features of this system to note is thatall analysis is carried out in the liquid phase within anenclosed chamber on disc, and that the analysis of the

biologic sample is carried out concurrently withoptical tracking of information features on disc.The BCD system is the testing platform of choice Fit contains all of the necessary elements.

K Centrifugation capability (variation in speeds andtiming enable sample processing within micro-fluidic chambers).

K The laser serves as the analytical tool used to readthe test results. The diagram in Figure 2 depictsthe basics of laser optics used for diagnostics. Thisis the ‘heart’ of the BTI platform.

K Software included on each BCD disc sends testdata securely and instantaneously via the Internetto a central data repository and authorizedindividuals who need the information.

Disc readerThe optical disc drive is fast becoming more thanjust a data storage and retrieval device. It is asophisticated laser-scanning device with inherentcapabilities in:

K physical process/measurementK image recognitionK laser scanning microscopy.

Just like a scanning microscope can be used tocharacterize microscopic objects, the optical disc driveis designed to identify micrometer-sized features at arate of a million per second. This feature can be used as

FIGURE 1. Micro-array technology in the BCD disc system. Clockwise from top right: atomic force microscopic imageof probe-captured DNA in the spot zone; three-dimensional presentation of DNA array data as analysed in the reader;amicro-array grid analysis screen of a BCD disc; a BCD discwith amicro-array cassette; and a readerdevice for readingBCD discs.

TABLE1. Principalof the technology: highlights

K The optical disc drive is a sophisticated laser-scanningmicroscope.

K The optical disc drive is also a centrifuge.K The CD (DVD,HD-DVD) is a biocompatible ‘solid phase’,

which can substitute for standard consumablesincluding slides, micro-wells, centrifuge tubes.

K Real-timeanalysispossible: forexample, a1mm2

micro-array canbe scanned in 20^30 swith a totaldata reduction time of 5 min.A customalgorithmperforms interpretations in real time.The image showsanoverall scheme of BTImicro-arraydesignandanalysis.

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indicated in Figure 2 to identify objects by theirelectronic signatures, and even to quantitate thepresence of these signatures within a given field.

Micro£uidicsA key feature to adapting assays to the disc isnanotechnology (i.e. the miniaturization of systemsthrough nanofabrication processes). This is done insuch a way that assays can be scaled down withoutsacrificing performance. Nanotechnology also lowerstest cost by reducing reagent requirements. The BCDdisc utilizes systems of microchannels to deliver smallbut exact amounts of reagents and sample to theappropriate zones on the disc. In essence, themicrofluidic engineering is married with the high-resolution, optical capabilities of the CD/DVD read-er. Fluid flow has to be controlled to provide specialconditions where mixing is desired, or uniform flowover assay zones where homogeneous assay perfor-mance must be assured. As indicated earlier, the driveacts as a centrifuge, controlling sequencing of fluidaliquots into channels and flow rates over assayzones, etc., with highly calculated accuracy.

Micro-array technologyMicro-array technology is a very important platformtool, and its application is just as important inimmunoassays as it is in genetic analysis (Joos et al.,2000; Kim et al., 2000). Micro-arrays can provide

huge amounts of biological data, which can beapplied to answer many questions at one time. Figure1 provides an indication of how arrays are integratedinto the BCD disc. Arrays of 30–75 mm diameterspots can be densely grouped on the disc to readilyachieve from 150 to 1500 spots/mm2 (Kiddo et al.,2000). Several formats of data analysis can be used.Figure 3 shows a simple array of three spots, withwhich only the determination of the presence orabsence of the probed sequence is desired. The arrayspot is analysed for the overall concentration (ordensity) of beads bound to hybridized complexes.Further a quantitative relationship of reporter beadsand the amount of probed sequence present can beassessed, and the copy number can be defined inrelation to internal controls within the testing system.This provides both a qualitative and a quantitativeformat for data analysis.

The principle for generation of signals for the formatgiven in Figure 3 is shown schematically in Figure 4.The capture probe zone, in which target DNA iscaptured, can be designed into a microline format (forgenetic fingerprint visualization and display) or intomicrodot arrays (for analysis by the reader). Further-more, the multiple probe combinations can be usedwithin the same capture spot. As previously noted,each object provides a unique electronic signature(Figure 2). Because of this attribute, reporter micro-spheres of different characteristics (size, light transmis-sive or light absorbing, etc.) can be used together. Each

FIGURE 2. Schematic of a BCD disc readerand a picture of the internal drive components.Reportermicrospheres on thedisc result in electronic signatures to be generated that are characteristic of the microspheres. Such signatures can beutilized to provide eitherqualitative orquantitative results.

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reporter can be coated with different receptors thatcorrespond with specific ligands, which in turn arepresent on the amplified target DNA (e.g. biotinylatedtarget DNA will attract strepavidin-coated micro-spheres, while digoxigenin-labelled target DNA wouldattract microspheres labelled with anti-digoxigenin).Such combinations become useful in analysis of mixedallotypes or for quantitating target DNA againstinternal control DNA copies.

By integrating previously separate steps of stan-dard assay formats into a seamless, simplifiedprocess, this new technology enables rapid, inexpen-sive and unambiguous evaluation of clinical samples.

What previously required many steps and up to24 h before a physician could be given test results, will

now take only a few steps and just minutes. The keyadvantage of the BTI system is that it dramaticallysimplifies the testing process and makes it moreconvenient by replacing complex (and expensive) testequipment with an ordinary PC and a CD/DVDreader. Thus, testing can be done at the point of careas easily as it could be done in a laboratory setting.

APPLICATIONS IN INFECTIOUSDISEASE

An area of practical application of the BCD disc is inthe diagnostics and characterization of infectiousdisease. The disc can be utilized to perform immu-noassays for detection of antibodies or antigens thatmight be associated with infection by a particular

Sample Addition Port Fluidic Channel To Disc Margin

FIGURE 3. A qualitative result for a simple triplet array.Biotinylated target DNA is added and hybridized to capture probe.Strepavidin-Reporter microspheres are added.The disc is spun for 5min, at 3,000 rpm to remove free microspheres.Thereader thenanalyses event signatures in the capture zone.

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pathogen. But for this discussion, utilization of thedisc for detection DNA or RNA of the pathogen willbe emphasized. Based on similar reporter systems asthose already described, micro-arrays for proprietarytarget sequences are being prepared to identifyspecific pathogens. The process begins with a sample(e.g. body fluids such as blood, urine, saliva or milk,

tissue, etc.), followed by extraction of nucleic acid,amplification of targets and, finally, hybridizationand detection using micro-arrays (the last step beingperformed in the BCD disc). Diagnostics currentlybeing developed will enable multiplex identificationof specific pathogen types such as Mycobacteriumparatuberculosis, Staphylococcus aureus, coliformorganisms, streptococcal species, Salmonellae, Bru-cellae, Mycobacterium bovis, Campylobacter, HIV,hepatitis type B and type C, and other pathogens.

One such example would be for use with brucellosisinfections. Brucellosis, a disease of humans andanimals, has both health and economic importancein many developing countries (Nicoltti, 1980; Achaand Szyfres, 1987). Six species of Brucella arecurrently known to cause brucellosis: Brucella abor-tus, Brucella suis, Brucella melitensis, Brucella ovis,Brucella neotomae, and Brucella canis (Nicoltti, 1980;Acha and Szyfres, 1987). Infection begins as septice-mia with sudden insidious onset characterized bycontinued, intermittent or irregular fever (Acha andSzyfres, 1987). Symptomatology is similar to that ofother febrile illnesses, but with a marked musculo-skeletal involvement (severe fatigue, aches and pains)(Acha and Szyfres, 1987). Severe complicationsinclude meningitis, encephalitis, spondylitis, suppura-tive arthritis, and vegetative endocarditis (Acha andSzyfres, 1987). The infection is treatable with tetra-

FIGURE 4. Use of reporter microspheres in signal analy-sis. Capture probes within the array spot hybridize to thetarget DNA, which is biotinylated (free biotinylated primersare not complementary to the capture probe sequences).Streptavidin-coated microspheres then bind to the spotzones in which the target DNA is hybridized. Unbound mi-crospheres are removed through spinning the disc.

Base Pairs

300

200

100

B. melitensis

B. melitensis

B. suis

B. suis

B. abortis

B. abortis

PCR generated amplicons stained with ethidium bromide

Multiplexed (3 species) Probes

FIGURE 5. Multiplexampli¢cationanddetection: agarosegeland CDreader results.Gel results showmultiplex-ampli¢edDNA from three species of Brucella.These productswere thenhybridized to probe capture zones on the disc and analysed(after addition of reporter microspheres) for the presence of target DNA.Each capture zone provides speci¢c detection ofDNAof the species of interest or in the presence of DNAofanyof the species of interest (multiplexedprobezone).




cyclines, but can become established chronically insome cases.

Figure 5 indicates how amplified DNA fromdifferent species of Brucella can be detected usingthe BCD disc. The region amplified codes for aportion of 16s RNA (Dorsch et al., 1989; Fox et al.,1998). One primer (forward) is common for allspecies, while the second primer (reverse) differs foreach species. With this design, the amplified DNAproduct from each pathogen differed in number ofbase pairs, but also each product had sequences thatare specific for that pathogen. The results show thatDNA from each organism could be detected usingarray spots with probes specific for that species, orthe presence of DNA from any of these organisms ina sample could be detected in an array spot contain-ing probes for all three organisms (a multiplexedprobe spot or zone). The detection process is highlyspecific, since removal of B. suis DNA (or DNA ofanother species) from the test sample results in anegative result from the array analysis for thatorganism (data not shown). It should be noted fromthis that the specific pathogen could be identifiedusing species-specific probe spots or, if desired toscreen for multiple potential pathogens, arrays ofmultiplexed probe spots could be used to searchthrough to multiple possible pathogens. A similardetection format has been used for detection of B.abortus and M. bovis in milk collected from infectedherds of cattle (Sreevatsan et al., 2000). That assaysensitivity was found to detect as few as 10–100genomic DNA equivalents per milliliter of specimen(Sreevatsan et al., 2000).

Viral load analysis is a standard of practicefor HIV-infected and hepatitis C virus-infectedpatients currently, not only in monitoring clinicalstatus of patients, but also (and more importantly)in monitoring efficacy of drugs on patients (Zeinet al., 1996; Bell et al., 1997). Since the technologyrequires operators with skill and experience, andvirtually mandates that molecular biologists managelaboratories performing these assays, testing islimited to those hospitals and clinical laboratoriesthat can provide this type of support (Krajden, 1995).Adaptation to the BCD disc platform for the virusdetection (and quantifying viral RNA copiesper milliliter of plasma) is currently underway.A third infectious disease, molecular applicationunder study for the BCD disc utilization is genotypicfingerprinting for drug resistance in HIV, hepatitis Cvirus and Mycobacterium tuberculosis. These lastapplications entail the utilization of extensive arraysthat probe for the specific known mutations ofinterest.

APPLICATIONS IN NEUROGENOMICSANDPHARMACOGENOMICS

To most in the field of human genomics, the post-genomic era is the close of just the beginning. The nextstep is to mine through the millions of pieces ofinformation that comprise the genome and make senseof its composition. In so doing, a better understandingof gene associations with disease, drug response andother phenotypic expression will be gained for use byphysicians and pharmaceutical companies.

It is now estimated that the human genome iscomposed of about 50 000–90 000 genes, but thatutilization of those genes results in more than 1million proteins (of which fewer than 8000 genefunctions are known) (Kennedy, 2000; Roest et al.,2000; Venter et al., 2001). One of the first tasks in thisnew era is to catalogue the most common types ofgenetic differences. Exons and regulatory sequencesconstitute only about 1–3% of the genome (Venteret al., 2001). About 75% of the genome lacks truegene composition, as it consists mainly of random,nonsense and repetitive sequence regions (Venteret al., 2001). Single nucleotide polymorphisms (SNPs)occur at about one in every 1250 base pairs (clusteredor dispersed and in coding or non-coding regions),and constitute about 80% of all sequence-defineddifferences in populations (Altshuler et al., 2000;Sachidanandam et al., 2001; Venter et al., 2001). Theshort tandem repeats, usually of five to nine basesequence repeats, are also present in the genome atthe frequency of about one in 3 million base pairs(Edwards et al., 1991, 1992; Warne et al., 1991;Kupferschmid et al., 1999).

While other technologies may currently be requiredfor analysing genotypic differences resulting fromshort tandem repeats, the BCD disc technologies arevery useful for detecting SNPs and many knowngenetic deletions and insertions. As already notedwith the previous examples, specific genetic sequencescan be detected using arrays of various compositionson the BCD disc. It is now possible to identifydisease-validated targets and use them to aid in bothdiagnostic and therapeutic decisions. To illustratethis, the rapidly unfolding advances in two importantareas of gene discovery will be discussed: pharmaco-genomics and neurogenomics.

Pharmacogenomics is a segment of genome re-search that emphasizes discovery of genes that affecthow drugs work in different individuals (Weber, 1997;Kleyn and Wesell, 1998). Population differences anddisease associations with genetic variation are criticalfor this process, as pharmacogenomics is an efforttoward a systematic approach to elucidating gene

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function and validating drug targets (Miller et al.,1997; Ingelman-Sundberd, 1998; Kleyn and Vesell,1998). Arrays can be used to provide gene expressionprofiles that help define the effect of therapeuticcompounds of relevant genes. Genetic variations canaffect expression patterns, can impair or enhance drugeffects and can even contribute to drug toxicity (Milleret al., 1997; Ingelman-Sundberd, 1998).

Several examples of genotypic polymorphisms andtheir effect on drug utility are presented in Table 2. Itis estimated that only 30–60% of patients with thesame disease will give an effective response to manyof the commonly used drugs (Shi et al., 2001). But avariable number will also have a range of side effects.Both efficacy and adverse reactions have foundationsin genetic make-up. CYP3A4, for example, is knownto affect the metabolism of a large number of drugs(Evans and Relling, 1999). Several altering mutationsare known, including a common promoter poly-morphism (Evans and Relling, 1999). Anotherexample is ApoE4, an apolipoprotein E locusallotype that not only correlates with elevated riskfor Alzheimer’s disease, but serves as a negativepredictive marker for response to cholinesteraseinhibitor treatment (Farlow et al., 1996, 1998). Manymutations known to affect drug activity are SNPs.About 2–10% of the population are homozygous fornon-functional CYP2D6 alleles (Evans and Relling,1999). This correlates with reduced codeine responseand less pain tolerance (Evans and Relling, 1999).Known CYP2D6 mutations include splice site muta-tions causing exon skipping, deletions and geneduplications, and early stop codon point mutations(Evans and Relling, 1999). Many diagnostic groupsare using or developing the array technology as aformat for looking at the various pharmacogenomicmarkers such as those indicated.

Another segment of genome research is neuroge-netics, which emphasizes exploring genetic variation

and the effects on neurologic diseases. Table 3illustrates (by the selected diseases listed) the diversityof genes affected or contributing to diseases withneurologic symptoms.

Similarities with the pharmacogenomic researchmay be noted. Several of the metabolic diseases,particularly Lesch–Nyhan syndrome and atypicalphenylketonuria (PKU) (Type II), appear to involvethe dopaminergic pathways in the brain (pathwaysinvolved in the pathogenesis of many other neurolo-gic and psychiatric diseases including Parkinson’sdisease and schizophrenia). Investigations into howthese in-born errors of metabolism produce seizuresand abnormal behaviour may provide insight intohow these same systems malfunction in other morewidespread diseases.

Mutational analysis can also reveal patients withhomocystinuria who are cobalamin (B6 vitamin)responsive or who are unresponsive. Responsive pa-tients usually have residual cystathionine b-synthetaseactivity, which can be further stimulated by large dosesof the B6 vitamin, while non-responsive patients havemutations that completely abolish the enzyme activity.

Some diseases are caused in a defect of a singlegene, yet the location of the mutation affects thetypes or degree of symptoms expressed, while otherdiseases may result from mutations in different genes.And again, for example, with Maple Syrup UrineDisease, the mutation may result in expression ofdifferent clinical symptoms, suggesting contributoryinfluences not well defined to date. Yet specificmutations identified in Table 3 are known.

The use of micro-arrays that identify specificmutations of a patient could greatly benefit theunderstanding of underlying effects and influences ofgene interactions. For example, the number ofpossible gene variants makes array technologypotentially very useful in prenatal genetic diagnosisof PKU. Over 70 different mutations of the gene have

TABLE 2. Genotypingdrugmetabolism: drugexamples anddruge¡ect alteringmarkers (Farlow et al.,1998; KleynandWesell,1998; Emilein et al.,1999; Evans and Relling,1999; Shiet al., 2001)

Drug Druge¡ect alteringmarkers

Acetaminophen CYP1A2,CYP2E1,CYP3A4 (genesof cytochrome P450 superfamily)Beta blockers CYP2D6 (Debrisoquinhydrolase)AntidepressantsAntipsychoticsNeuroleptics Dopamine receptors D2,D3,D4,D5Cholinergic agents ApoEFallotype E4(Alzheimer’s disease,Tourette syndrome)Dexamethasone Glutathione S-transferasesFYb1,Yb2 and Yb3Lovastatin CYP2B1/2,CYP3A1/2,CYP3A4Thiopurine Thiopurinemethyl transferase,CYP2D6




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https://www.researchgate.net/publication/12779865_Evans_WE_Relling_MV_Pharmacogenomics_translating_functional_genomics_into_rational_therapeutics_Science_286_487-491?el=1_x_8&enrichId=rgreq-1d5c5f20-5a1f-4790-8408-1da43add2418&enrichSource=Y292ZXJQYWdlOzExMDE0NTE4O0FTOjEwMzMxNTcxOTMyNzc1NkAxNDAxNjQzNzk1NjQ0




TABLE 3. Selected reviewof neurologic diseasesandgenetic features

Disease Symptoms/neurologicmanifestations

Mutations(chromosome site)

Array requirements(challenges)

Additional comments References

Alzheimer’s disease Mainlyold-age onset ofmemoryandpersonalityloss.Severe degenerationof brainwith proteinplaques (beta-amyloid)andneuro¢brillarytangles.Progressivedementia andmemoryimpairment with at leastone of: aphasia, apraxia,agnosia or lossofexecutive functioning

Amyloid beta A4precursor proteinAPP (21q21)

Presenilin1 (14q24.3)Presenilin 2 (1q31^q42)Apolipoprotein

type E (19q13.2)

12þmutations72þmutationsAt least sixmutationsFourallotypesFE4

allotype carries risk

Otherassociatedmarkers still underinvestigation.Apo4is considered to bea susceptibility riskmarker.Mutationsin APPor Presenilinsfound in 30^40% ofautosomaldominantAlzheimer’s diseaseand 5% of casesin general

Goate et al.,1991;Corder et al.,1993;Hutton et al.,1996;De Jonghe et al.,1999;Finckh et al., 2000;Nilsberth et al., 2000

Carbohydrate-de¢ciencyglyco-proteinsyndrome

Only type Ia hasprominent neurologicalmanifestations.Severepsychomotor retardation(‘£oppybaby’syndrome).Multi-organ involvement

Phosphomannomutase(16p13.3^p13.12)

230þmutations anddeletions

R141H,N216IandF119Lde¢ne 88%of cases.Commonmutationsnoted forScandinavians

Hagberg et al.,1993;Jaeken et al.,1993;Van SchaftingenandJaeken,1995; Matthijset al.,1997,1999, 2000;Bjursellet al.,1998,2000; Kjaergaardet al.,1998,1999;Kondo et al.,1999

Galactosemia Symptomatic triad:hepatomegaly, cataractsandmental retardationfailure to thrivewithincreased intracranialpressureand cerebraledema.De¢cient speechand language skills.Cerebralatrophyandwhitematter lesions

Galactose1-phosphateuridyltransferase(9p13)

130þmutations Q188Rand K285Nde¢ne 70% ofCaucasian cases(absent inJapanese).S134Lcharacteristicof 62% of blackethnic cases.Duartevariant (N314D/N314D) gives 50%reducedenzymeactivity.Los Angelesvariant of Duartegives increasedactivity

Hsia,1967; ReichardtandWoo,1990,1991;Schweitzer et al.,1993;Langley et al.,1997;Elsas and Lai,1998

Homocytinuria Thromboembolicdisease; multi-organsa¡ected.Glaucoma,

Cystathionine b-synthetase (21q22.3)

Array to address492mutations

Mostlymissensemutations; mostcommon I278T

Mudd et al.,1985;Visy et al.,1991;Tsaiet al.,1996;

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osteoporosis.Mentalretardation (IQ 50^70)but one-third havenormal IQ.Many (not all)with psychiatricdisturbances (depression,obsessive-compulsive,etc.)

(vitamin B6responsive) andG307S (vitamin B6non-responsive)

Chasse et al.,1997;Kraus et al.,1999

Hyperphenyl-alaninemia

Type1 [classicalphenylketonuria (PKU)]:centralnervous systeminsult withmentalretardation, epilepsy,myelination defects.Treatment ismainlydietary

Phenylalaninehydroxylase (12q34.1)

70þ haplotypesFsixmutationsmakeup 80%of PKU.For Caucasians,most eithera singlebase change in the12th intronora R408Wmissensemutation

Variousethnicpatternsnoted.Mutation correlateswith type, residualenzymeactivityandPKUseverity

Paine,1957; DiLellaet al.,1986; GuttlerandWoo,1986; Daigeret al., 1989; Hofman et al.,1989, 1991; Scriver et al.,1989; Konecki and Lichter-Konecki,1991; Degioanniet al.,1994; Kaufman,1999;Gri⁄ths et al., 2000

Type II (atypical PKU):neurologicaldefectmore severe than type I.SymptomslikeParkinson’s disease

Dihydropteridinereductase (4p15.31)leads to a dysfunctionof tetrahydrobipterin(BH4)

50þmutationsFmissense, nonsense,insertions anddeletions

Inadequate levels of BH4:(a) six PTPS de¢ciency:Parkinsonian-likesymptoms, hypotonia,swallowingdi⁄culties,neuropsychologicaldevelopmentimpariments, seizures.(b) GTPcyclohydrolasede¢ciency

6-pyruvovyltetrahydropterinsynthetase(11q22.3^q23.3)

GTPcyclohydrolase(14q22.1^q22.2)

10þmutations

Only six patientsreported to date

Strongethnicpatternsnoted

Hypertyrosinemia Type1: pain in extremities(peripheralneurologicalcrisis) with autonomicdysfunction andprogressive paralysis.Bizarre postures torelievepain.Convulsions.Normal IQ.Treatablewithmedication

Furarylacetoacetatehydrolase (15q23^q25)

26mutationsFmissense, nonsensesplicing defects

Genotypeandphenotypecorrelationnotwell de¢nedto date

Natt et al.,1986,1992;Mitchellet al.,1990;Hahn et al.,1995;Timmers and Grompe,1996; Louis and Tanguay,1997

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Type II: involvement of skin,eyeand centralnervoussystem.B50% of caseshavemental retardation;somemicrocephalyandseizures.Pain fromtyrosine crystals in eye,hyperkeratotic plaquesin skin

Tyrosineaminotransferase(16q22.1^q22.3)

Sevenpoint mutationsreported ^missensenonsenseand splicingdefects

Lesch^Nyhansyndrome

Profoundneurologicalinsult andmentalretardation.Hypotonia,spasticity, coreoathroidmovements and self-mutilation.Compulsiveaggressive behaviour.Generalbrain de¢ciencyin dopaminergic neurons.Infants, mostlymale,begin symptomsat3^6monthsof age

Hypoxanthine-guaninephosphoriboyltransferase(Xq26^q27.2)

60þmutationsFmissense, nonsense,insertions and deletions

11alleles alsonotedfora CTATrepeat inintronbetweenexon 3 andexon 4

Wilson et al.,1983;Gibbs and Caskey,1987;Fujimoriet al.,1988;Davidson et al.,1989;Renwicket al.,1995

Maple syrup urinedisease(branched chainamino aciddisease)

Five clinical types:1.Classic (most common):

neurologic deteriorationof neonate.Seizures,cerebraledemas,deaths.o2% normalBCKADactivity

2. Intermediate (rare):older infants.Seizures,orepisodic keto acidosis,acutemetabolicdecompensationandencephalopathy, variablemental retardation.3^30% normal BCKADactivity

Molecular types:Branch chain a-keto

acid dehydrogenase^BCDAD complex

Type1A (classic andintermediate):a-chain of E1protein(19q13.1^q13.2) ofBCKAD complex

Sicmutations Y393Naccountsforalmost allMennonitepatients

Dancis et al.,1967;Wong et al.,1972;Fekete et al.,1989;Chuang et al.,1991;Fisher et al.,1991a,1991b;Herring et al.,1991;Nobukuniet al.,1991;Scherer et al.,1991;Zneimer et al.,1991;Liu et al.,1993; Chuangand Shih,1995; Pateland Harris,1995;Hong et al.,1996;Elpeleg et al.,1997

TABLE 3. [Continued]

Disease Symptoms/neurologicmanifestations

Mutations(chromosome site)

Array requirements(challenges)

Additional comments References

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been identified (Guttler and Woo, 1986; Scriver et al.,1989; Konecki and Lichter-Konecki, 1991), and six ofthese account for over 80% of PKU (Konecki andLichter-Konecki, 1991). Ethnic distributions of mu-tations have been shown in the Chinese, YemeniteJewish, and Afro-American populations (Daiger etal., 1989; Hofman et al., 1991; Degioanni et al.,1994). There is also good correlation between thetype of mutation, the amount of residual enzymeactivity, and the severity of the PKU (Daiger et al.,1989; Hofman et al., 1991; Degioanni et al., 1994).Future diagnostic tests should detect most possiblemutations in the phenylalanine hydroxylase gene andprovide phenotype/genotype correlations strong en-ough to predict which mutations will lead to a severePKU phenotype. Until that can be done, diagnosiswill continue to depend on screening newborn bloodfor phenylalanine levels.

Molecular diagnostics may become more routineas these technologies become available. In thisrespect, while numerous mutations are described foreach disease, because many are defined, arrays can bedeveloped to screen for these mutations. In so doing,a disease mutation could be detected in most cases,with only a small frequency not identified due to thepresence of a new mutation.

THECHANGES IN PROGRESS

The technological advances and molecular applica-tions, which we have noted, hold the potential toallow physicians and researchers to diagnose orfurther characterize the complexities of many dis-eases. Furthermore, these advances may soon aid intherapeutics: first, in the design of medications, basedon patient genotypes, that are more effective and lesstoxic; and second, by providing the medical practicewith better tools for therapeutic response assess-ments. It is anticipated that much of the guessworkcurrently associated with patient care will be reduced.

Acknowledgements

The authors would like to acknowledge the efforts of VitoDelveccio in providing sequence information and samplesfor the work in Brucellae speciation. Special thanks alsoto Ramon Valencia and Ryan McCrae for their con-tributions to the studies presented in this publication.

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