CA2398058A1 - Gene chip for neonate screening - Google Patents

Abstract

The invention relates to a nucleotide support containing a selection of oligonucleotide sequences for detecting reference sequences which are releva nt for at least two phenotypes, or their functionally characterized mutations. In said nucleotide support, the oligonucleotide sequences are identical or complementary to the reference sequences.

Description

~!I f "Gene chip for Neonate Screening The invention pertains to a nucleotide carrier for combined oligonucleotides with a selection of oligonucleotides for identifying specific gene sequences. It Priority of the German patent application 100 02 446.7 is claimed, which is hereby fully incorporated by reference.
Hybridising techniques, i.e. the targeted hybridisation of two complementary nucleic acid strands, form an essential part of various methods in molecular biology. These techniques are applied in order to detect single genes or parts thereof in a preparation of genomic DNA
or the transcription product of a gene (mRNA). [Sambrook J, Fritsch EF, Maniatis T.
Molecular cloning: A laboratory manual. 2nd ed., vol. 2, 1989, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 13.96-97; Constanzi C, Gillespie G: Fast blots:
immobilization of DNA and RNA from cells. In: Guide to molecular cloning techniques.
Edited by Berger SR and Kimmel AR, Academic Press Inc., San Diego: Methods in Enzymology 1987, 152: p582-87; Schena M et al.: Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995;270:
p467-470].
One of the most important applications of hybridisation is the investigation on mutations in genes [Ausubel FM, Brent R, Kingston RE, Moore DE, Seidman JG, Smith JA, Struhl K.
(Hrsg.) Current protocols in molecular biology, 1998, John Wiley & Sons].
Usually hybridisation is combined with a detection and a subsequent identification of a nucleic acid sequence. For this purpose one nucleic acid strand is labelled e.g. with coloured probes, radioactivity, or chemiluminescent or flourescent molecules, whereas the iil 1 I

second strand is bound to a solid phase. In the well known southern blots for DNA analysis or northern blots for RNA analysis, the solid phase often consists of a nitrocellulose- or a nylon membrane.
In these common blot-systems, genomic DNA or RNA - the so-called target sequences - are electrophoretically separated on an agarose jelly, transferred onto the nitrocellulose- or nylon membrane and hybridised with a known DNA- or RNA-sequence used as a probe.
Hybridisation is detected by prior labelling of the probe and quantified where appropriate.
These methods are characterised in a high specificity. However, the disadvantage, that the conduction of a blotting procedure is very labour-intensive and time-consuming and that only a few sequences can be investigated in one stage remains.
More recently, hybridisation of a probe and a target has been accomplished by the so-called gene chips (Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H and Brown EL. Expression Moniforing by Hybridization to High-Density Oligonucleotide Arrays. Nature Biotechnology.
14:1675-1680, 1996; Wodicka L, Dong H, Mittmann M, Ho MH, and Lockhart DJ. Genome-Wide Expression Monitoring in Saccharomyces Cerevisiae. Nature Biotechnology.
15:1359-1367,1997]. Gene chips consist of a solid carrier e.g. made of plastic or glass, onto which up to several thousand oligonucleotides can be fixed as probes.
Consequently, the surface of a chip which is limited to a dimension of some square centimetres is covered with a "lawn" of oligonucleotides, to which complementary nucleic acid sequences of a DNA- or RNA-probe can hybridise. Prior to hybridisation, the target sequences are labelled in order to enable the detection of hybridisation.
The use of gene chip allows for the investigation of several thousands of different sequences simultaneously and thus represents an improvement in comparison to common blot techniques. Furthermore the hybridisation on a chip can be analysed by means of a scanner, enabling an extensive automating of the procedure.
Several different techniques have been developed for the manufacture of gene chips or genetically combinatorial investigation devices, e.g. lithographic methods, sieve printing or reaction channel methods, electrochemicall-synthetical procedures, ink-jet systems, micropin-methods or open capillary tips. Another known technique applies micro beads ni [Lackner KJ et al. Multiplex DNA- and RNA-Analyse an fluoreszenten Microbeads als Alternative zum DNA-Array. Statusseminar Chiptechnologie fur DNA-Diagnostik and Sequenzanalyse in Deutschland. DECHEMA 1999].
Mutations in genes can also be identified by sequencing the genetic information (Sanger et al., Proc. Natl. Acad. Sci. U.S. 74:5463-5467, in 1977). Since sequencing of nucleic acids in general is very laborious in practice mutations are mostly detected indirectly, i.e. by investigating their biochemical and/or physiological properties by using biochemical (e.g.
enzyme-based immuno-essays), analytical (e.g. high performance liquid chromatography, gas chromatography, mass spectrometry) or bacteriological (so-called C3uthrie growth inhibition test) methods.
This approach encompasses the disadvantage, that mutations can be detected solely after the expression of the respective metabolic diseases or pathological syndromes.
Consequently, possible therapeutic measures can be chosen and applied only at a late stage, in which irreversible damages might already have occurred.
In order to prevent the development of pathological symptoms, the time lag between the identification of the genetic defect and the onset of a therapy or of preventive measures should be reduced to a minimum. This may be accomplished by detecting the mutation directly in the nucleic acid, if possible already in the patient's childhood.
Besides to sequencing, genetic aberrations can also be identified by the above mentioned hybridisation techniques. Hybridisation offers the advantage, that gene chips might be used as well as automated evaluation and analysis systems.
US patent 5, 837, 832 describes a gene chip for screening of known sequences for possible mutations. First a sequence complementary to the targeted "reference sequence"
- the so-called wild type sequence - is synthesised in subsections each comprising preferably 12 to 18 bases. Furthermore sequences are synthesised, which differ from the wild type sequences only in a single base. The total number of these so-called substitution sequences is determined by the theoretically possible number of single base exchanges.

a Subsequently, the sequences are transferred to the chip in a defined pattern preferably in blocks each comprising five rows located side by side. These blocks each comprise one wild type sequence and in parallel thereto three substitution sequences with one base exchange. The fifth sequence is identical to the wildtype sequence. Thus, five rows and five sequences are necessary to represent all possible base exchanges of a single nucleotide.
In US patent 5 837 832 human genes are analysed for mutations according to this scheme, for example specific exons of the CFTR gene which might correlate to cystic fibrosis or the tumor suppressor gene p53, which might be associated with cancerous diseases.
This approach is disadvantageous since even in the case of rather short genes already some 10.000 or 100.000 different sequences have to be attached to the chip as potential hybridisation targets in order to include the whole range of possible mutations. In case of longer sequences of a gene or gene section under investigation or in case of a simultaneous examination of two or more genes or gene sections, the number of hybridisation sites necessary for a reasonable screening far exceeds the manufacture possibilities of a gene chip.
In order to investigate the approximately 40.000 human genes according with this method would require a gene chip with at least 4 x 108 binding sites. However, with todays' standards and even with future technical improvements to be expected, the production of a chip that would meet these requirements is not possible. In consequence a complete and early dated investigation of genetic defects is practically not possible.
Thus, the underlying problem of the invention is to provide a nucleotide carrier for combined oligonucleotides and a selection of sequences for such a nucleotide carrier or comparable genetically combined investigation material, especially for neonate screening enabling the examination of the genetic disposition of a defined combination of disease associated genes or gene sections (reference sequences).
This problem is solved by means of the independent claims. Favourable aspects thereof are subject of the dependent claims.

lil ~'i More specifically, the problem is solved by a nucleotide carrier (subsequently also "gene chip") with a selection of oligonucleotides with functionally characterised mutations known to cause specific phenotypes. These oligonucleotides can be identical or complementary to the reference sequences.
According to the invention the mutated oligonucleotides are carefully selected and combined from the spectrum of all theoretically possible mutations of the reference sequences sections.
These mutations are combined on the chip with at least one oligonucleotide causing a second phenotype.
The term "phenotype" hereby is to be understood in a broad sense and refers to all characteristics and consequences associated with a selected reference sequence or being detectable by detecting this sequence.
The phenotype can represent a body function, a physiological function, e.g. a metabolic disturbance or a genetically based disposition for a disease in the medical definition, e.g. a certain form of cancer.
The chip according to the invention offers the essential advantage, that only a limited number of hybridisation positions is required and thus enabling the reasonable application of the gene chip and the subsequent analyses in the medical practice.
A further advantage of the gene chip according to the invention is based on providing a fast, cheap and reliable investigation method with a sensitivity exceeding commonly used physiological methods.
An especially preferred embodiment according to the invention enables the control of genetic conditions in the prenatal and neonatal period.
In a screening programme for neonates a large number of genetically based metabolic diseases can be detected by using only a small blood volume. According to the invention, a III

selection of oligonucleotides for at least two or more of the following diseases can be combined on the chip:
Phenylketonuria, maple syrup urine disease, galactosaemia, homocystinuria, biotinidase deficiency, middle branched acyl-CoA-dehydrogenase-deficiency, familiar hypercholesterinaemia, familiar defect of apolipoprotein-B, cystic fibrosis, Marfan-syndrome, Smith-Lemli-Opitz-syndrome, adrenogenitale syndrome.
Espeaally preferred is a chip carrying oligonucleotides for the syndromes of "phenylketonuria" and "galactosaemia". Furthermore, this is combined with oligonucleotides for the "biotinidase deficiency".
These diseases are caused by mutations in a total of 21 definite genes, comprising about 150.000 nucleotides, when functionally relevant regulatory sequences are included. Since each nucleotide can be present as adenine, guanine, cytosine or thymine and also insertions, deletions or inversions have to be considered, the number of possible mutants amounts to about 900.000. In case of additionally investigating complex mutations like e.g.
small indels (insertionldeletion), the mutations easily reach a number of several millions:
A gene chip according to present the state of the art would therefore need several millions of corresponding hybridisation positions. Such a vast number of sequences cannot be reasonably placed or analysed on a chip.
By the oligonucleotide selection according to the invention, the sequences on the gene chip required for disease detection can be reduced to about 5.000. In this way, a simultaneous detection of mutations is possible on a single gene chip.
It is also possible to select between the oligonucleotides necessary for detection of the above cited diseases or to diagnose only one or a few diseases.
The diseases diagnosed by this preferred embodiment represent a careful selection of the most relevant genetically based human diseases. Their selection was further based on therapeutic options and prevalence within the population. Additionally they mainly go back ru to already characterised mutations. These functionally characterised mutations were combined and together cause 90 to 95% of the respective diseases.
According to this principle, also further diseases with an investigated genetic base can be identified alone or in combination.
It is also possible to identify or detect diseases or phenotypes in other organisms, e.g. in animals.
The invention is described by the following example of a selection of diseases:
The gene chip according to the invention and the selection according to the invention is based on oligonucleotides for the detection of reference sequences -comprising also the complementary sequences - that are located in 21 genes in the human genome. In particular, the following sequences are selected, which are indicated with the genebank code of the National Center for Biotechnology Information (NCB!). The respective diseases to be diagnosed are indicated in brackets:
1. Phenylalaninehydroxylase, PAH (diagnosis of phenylketonuria) [GenBank-numbers:
K03020, NM 000277, L47726, U49897].
2. Quinoid Dehydropteridin Reductase, QDPR (diagnosis of phenylketonuria) [GenBank-numbers: NM 000320, M16447, X04882].
3. Mitochondrial branched-chained alpha-ketoaciddehydrogenase, a-peptide, BCKDHA
(diagnosis of maple syrup urine disease) [GenBank-number: 214093].
4. Mitochondrial branched-chained alpha-ketoaciddehydrogenase, p-peptide, BCKDHB
(diagnosis of maple syrup urine disease) [GenBank-number: M55575].
5. Branched-chained dihydrolipoamid-transacylase, DBT (diagnosis of maple syrup urine disease) [GenBank-number: X66785].
6. Galactose-1-phosphat uridyltransferase, GALT (diagnosis of galactosaemia) [GenBank-numbers: M60091, NM 000155; L46354 to 46365, L46691 to 46724).
7. Galactokinase, GALK1 (diagnosis of galactosaemia) [GenBank-numbers: NM
002044, NM 000154, L76927, U26401, M84443].

8. UDP-Galactose-4-epimerase, GALE (diagnosis of galactosaemia ) [GenBank-number:

I71 f i L41668] (diagnosis of galactosaemia ).

9. Cystathion-beta-synthase, CBS [GenBank-numbers: NM 000071, L14577, X98810 to X98823, X88562, X87815, X87816, X91910].

10. Methyltetrahydrofolat-L-homocysteine-S-methyltransferase, MTR (diagnosis of homocystinuria) [GenBank-number: AF025794, NM 002454].

11. 5,10-Methylenetetrahydrofolat reductase (NADPH), MTHFR (diagnosis of homocystinuria) [GenBank-numbers: 009806, AF105988 to AF105998].

12. Biotinidase, BTD (diagnosis of a biotinidase deficiency) [GenBank-numbers:
063274, 003274].

13. Medium chain acyl-CoA-dehydrogenase, ACADM (Diagnosis of a medium chain acyl-CoA-dehydrogenase-deficiency) [GenBank-numbers: M16827, M91422 to M91432, NM
000016].

14. Low-density-lipoprotein receptor, LDLR (diagnosis of familiar hyper-cholesterinemia) [GenBank-numbers: NM 000527, L00336 to 00352, L29401].

15. Apolipoprotein B (diagnosis of a familiar defect of apolipoprotein B) [GenBank-numbers:
X04506, M14162].

16. "Cystic fibrosis transmembrane conductance regulator", CFTR (diagnosis of cystic fibrosis) [GenBank-numbers: NM 000492, M55131].

17. Fibrillin-1, FBN1 (diagnosis of a Marian-syndrome) [GenBank-numbers: NM
000138, X63556, L13923].

18. "Latent-transforming growth factor", beta-binding protein-2, LTBP2 (diagnosis of a Marian-Syndrome) [GenBank-number: Z37976).

19. Delta-7-Sterolreductase, DHCR7 (diagnosis of a Smith-Lemli-Opitz-syndrome) [GenBank-number: AF034544].

20. Sterol 21-hydroxylase, CYP21 (diagnosis of an Adrenogenital syndrome) [GenBank-numbers: M26856, M13935, M13936].

21. Sterol 17-hydroxlase, CYP17 (diagnosis of an Adrenogenital syndrome) [GenBank-numbers: NM 000102, M14564, M31146].
The gene chip according to the invention can also comprise oligonucleotide sequences identical or complementary to the respective sections of the reference sequences.
Furthermore, oligonucleotides comprising functionally characterised mutations or ~a i complementary sequences can be used. The mutations can be base substitutions, insertions and deletions. Possible further mutations of complex character might be inversions and indels (as insertionsldeletions).
The length of the oligonucleotides on the gene chip can range from 16 to 25 nucleotides.
Preferably, 15-mers to 18-mers are used.
If the oligonucleotides identical to the sections on the reference sequence are transferred onto the gene chip, the respective complementary sequences can be synthesised.
This synthesis is performed in the course of the labelling reaction necessary for the subsequent detection of successful hybridisation.
The sequences attached to the gene chip can be DNA- as well as RNA-sequences.
Also nucleotides with uracil or base analogues can be used.
The gene chip according to the invention offers the advantageous possibility to be extended to future sequenced and functionally characterised oligonucleotides.
The invention is further explained by the following example.
Example 1:
DNA-chip for detection of phenylketonuria (PKU) Oligonucleotides complementary to sequences of the reference sequences cited in table 1, are attached to a suitable carrier by means of standard techniques as being described e.g.
in German patent 196 12 356 or US- patent 5 837 832. This carrier preferably consists of gold-coated glass. The oligonucleotides have each a length of 15 nucleotides.
In certain cases also sequences with 16 to 25 nucleotides are possible.

.n The carrier is devided into a multitude of fields, each of which contains one sequence with only one mutation. Therein the sequences are selected and positioned each in a way, that the mutation has a most central position within the reference sequence section.
According to the invention the diagnosis of the mutations in the phenylalanine-hydroxylase-gene (PHA-gene) leading to phenylketonuria requires a total of only 548 fields. Hereby, the field 1.1 e.g. carries the sequence CAGTGGACATGCTGG complementary to reference sequence section CCAGCATGTCCACTG (table 1, line 1, column 2) and field 1.2 the respective mutated oligonucleotide CAGTGGATATGCTGG complementary to the mutated reference sequence section CCAGCATATCCACTG (table 1, line 1, column 3;
mutation underlined).
For the fields 2.1 and 2.2 and all further fields, the sequence information of line 2 in table 1 is continually used in the same manner.
Table 1: List of the nucleic acid sections, of which the corresponding complementary sequences are attached to the chip. To keep the list readable, only the central part of the 15- to 25-mers is shown. The sequence parts which are not presented are complementary to the reference sequence. The position relative to the reference sequence is to be seen from the indicated codon number.
An oligonucleotide complementary to the mutated reference sequence section is attached to the carrier each at an exactly defined position. The nucleotides differing from the reference sequence are to be found in line 3 of the table.
The sequence information listed in this table refers to a reference sequence found under GenBank-number U49897. The selected mutations comprise missense- or nonsense-mutations (table 1A) as well as splice variants (table 1 B), deletions (tables 1 C and 1 E), insertions (table 1 D), indels and complex rearrangements.
A) Missense- or nonsense-mutations Codon-No. reference mutated sequenceamino acid-(reference sequence exchange se uence 1 ATGt ATA Met-Ile 1 cATG GTG Met-Val 20 aCAG TAG Gln-Term 39 TTCt TTG Phe-Leu 40 TCA TTA Ser-Leu 41 aCTC TTC Leu-Phe 46 tGGT AGT GI -Ser 47 GCA GTA Ala-Val 48 TTG TCG Leu-Ser 52 TTG TCG Leu-Ser 53 CGC CAC Ar -His 55 TTT TTG Phe-Leu 56 GAG GAT GI u-As 63 ACC CCC Thr-Pro 64 cCAC AAC H is-As n 65 ATT AAT I le-Asn 65 ATT ACT I le-Thr 67 aTCT CCT Ser-Pro 68 AGAc AGT Ar -Ser 70 tTCT CCT Ser-Pro 77 TAT TAG T r-Term 81 cACC CCC 'Thr-Pro 84 GAT TAT As -T r 87 AGCc AGA Ser-Ar 87 tAGC CGC Ser-Ar 89 CCT TCT Pro-Ser 92 ACA ATA 'Thr-Ile 94 - ~ ATC ~ AGC ~ Ile-Ser 98 TTG TCG Leu-Ser 104 GCC GAC Ala-As 111 aCGA TGA Ar -Term 120 TGG TAG Tr -Term 122 CCA CAA Pro-Gln 124 ACC ATC Thr-Ile 129 GAC GGC As -GI

132 GCC GTC Ala-Val 143 GAT GGT As -GI

145 GAC GTC As -Val 147 cCCT TCT Pro-Ser 148 tGGT AGT GI -Ser 151 GAT GGT As -GI

155 CGT CAT Ar -His 158 CGG CAG Ar -Gln 158 aCGG TGG Ar -Tr 161 TTT TCT Phe-Ser 164 ATT ACT I le-Thr 165 tGCC ACC Ala-Thr 166 TACa TAA T r-Term 167 AAC ATC Asn-I 1e 171 GGG GCG GI -Ala 172 CAG TAG Gln-Term 173 CCC ACC PraThr 174 ATC ACC Ile-Thr 174 cATC GTC I le-Val 175 cCCT GCT Pro-Ala 176 CGA CTA Ar -Leu 176 tCGA TGA Ar -Term 177 aGTG CTG Val-Leu 178 GAA GGA Glu-GI

182 GAA GGA Glu-GI

187 TGG TAG Tr -Term 187 TGG TGC Tr -C s 187 aTGG CGG Tr -Ar 190 GTG GCG Val-Ala 194 CTG CCG Leu-Pro 201 cCAT TAT His-T r 203 TGC TAC C s-T r 204 TAT TAG T r-Term 206 TACa TAG T -Term 207 AAT AGT Asn-Ser 207 cAAT GAT Asn-As 211 tCCA ACA Pro-Thr 213 CTT CCT Leu-Pro 217 cTGT GGT C s-GI

218 GGC GTC GI -Val 221 GAA GGA Glu-GI

222 GAT GGT As -GI

224 ATT ACT I le-Thr 224 ATTc ATG I le-Met 225 CCC CGC Pro-Ar 225 tCCC ACC Pro-Thr 230 cGTT ATT Val-Ile 231 tTCT CCT Ser-Pro 232 tCAA TAA Gln-Term 238 cACT CCT Thr-Pro 239 GGT GCT GI -Ala 239 GGT ' GTT GI -Val 239 tGGT AGT GI -Ser 241 CGC CAC Ar -His 241 CGC CTC Ar -Leu 241 cCGC TGC Ar -C s 242 cCTC TTC Leu-Phe 243 CGA CAA Ar -Gln 243 cCGA TGA Ar -Term 244 CCT CTT Pro-Leu 245 GTG GAG Vai-Glu 245 GTG GCG Val-Ala 245 tGTG CTG Val-Leu 246 GCT GTT Ala-Val 247 GGC GTC GI -Val 248 CTG CCG Leu-Pro 249 CTT TTT Leu-Phe 252 CGG CAG Ar -Gln 252 tCGG GGG Ar -GI

252 tCGG TGG Ar -Tr 255 TTG TCG Leu-Ser 255 cTTG GTG Leu-Val 259 GCC GTC Ala-Val 261 CGA CAA Ar -Gln 261 CGA CCA Ar -Pro 261 cCGA TGA Ar -Term 263 TTCc TTG Phe-Leu 265 TGC TAC C s-T r 269 ATC AAC Ile-Asn 269 cATC CTC I le-Leu 270 AGA AAA Ar -L s 270 AGAc AGT Ar -Ser 272 tGGA TGA GI -Term 273 TCC TTC Ser-Phe 276 ATGt ATT Met-I 1e 276 -. ~TG -~ GTG - - Met-Val 277 TAT GAT T r-As 278 ACC AAC Thr-Asn 278 ACC ATC Thr-Ile 280 cGAA AAA Glu-L s 281 CCT CTT Pro-Leu 282 tGAC AAC As -Asn 283 ATC AAC Ile-Asn 283 cATC TTC Ile-Phe 285 cCAT TAT His-T

295 TCA TGA Ser-Term 297 CGC CAC Ar -His 297 tCGC TGC Ar -C s 299 TTT TGT Phe-C s 300 GCC GTC Ala-Val 300 tGCC TCC Ala-Ser 303 tTCC CCC Ser-Pro 306 aATT GTT Ile-Val 309 GCC GAC Ala-As 309 GCC GTC Ala-Val 311 CTG CCG Leu-Pro 314 CCT CAT Pro-His 320 AAGc AAC L s-Asn 322 GCC GGC Ala-GI

322 cGCC ACC Ala-Thr 325 TACt TAG T -Term 326 TGG TAG Tr -Term 327 TTTa TTG Phe-Leu 331 TTT TGT Phe-C s 331 TTT CTT Phe-Leu 333 CTC TTC Leu-Phe 336 aCAA TAA Gln-Term 338 aGAC TAC As -T r 341 aAAG TAG L s-Term 342 GCA ACA Ala-Thr 342 GCA CCA Ala-Pro 343 TAT TGT T -C s 344 tGGT AGT GI -Ser 345 tGCT ACT Ala-Thr 346 tGGG CGG GI -Ar 347 CTC TTC Leu-Phe 348 cCTG GTG Leu-Val 349 TCA TTA Ser-Leu 349 TCA CCA Ser-Pro 350 aTCC ACC Ser-Thr 352 tGGT CGT GI -Ar 355 aCAG TAG Gln-Term 356 TACt TAA T r-Term 356 TACt TAG T -Term 359 TCA TGA Ser-Term 360 aGAG TAG Glu-Term 362 CCA ACA Pro-Thr 366 CCC CAC Pro-His 367 cCTG GTG Leu-Val 371 AAG AGG L s-Ar 372 ACA TCA Thr-Ser 380 AGG ATG Thr-Met 386 TAT TGT T r-C s 387 tTAC CAC T r-His 388 cGTG ATG 'Jal-Met 388 cGTG CTG Val-Leu 390 GAG GGG Glu-GI

394 GAT GCT As -Ala ~n 394 tGAT CAT As -His 395 GCC GGC Ala-GI

395 tGCC CCC Ala-Pro 403 GCT GTT Ala-Val 407 aCCT TCT Pro-Ser 408 CGG CAG Ar -Gln 408 tCGG TGG Ar -Tr 413 CGC CCC Ar -Pro 413 tCGC AGC Ar -Ser 414 TAC TGC T -C s 415 cGAC AAC As Asn 417 aTAC GAC T -As 418 cACC CCC Thr-Pro 447 GCC GAC Ala-As Column 1 indicates the number of the codon affected by the mutation in the reference sequence. The last column shows the amino acid exchange in consequence of the mutation.
B) splice variants Intron-No. Type of the Position of Base-splice site the exchan a mutation 1 Donor +5 G-C

1 Don or +5 G-T

2 Acce for -13 T-G

2 Donor +1 G-A

2 Donor +5 G-A

2 Donor +5 G-C

2 Donor +5 G-T

2 Donor +6 T-G

3 Acce for -1 G-T

3 Acce for -6 T-A

4 Acce for -2 A-C

4 Acce for -5 C-G

4 Acce for -1 G-A

4 Donor +1 G-A

4 Donor +5 G-T

5 Donor _ +1 G-A

6 Donor -96 ~ A-G

- Acceptor -2 A-T

7 Donor +1 G-A

7 -- Doncr +5 G-A

Donor _ +3 ~ G
-C

_ 7 Donor +2 _ _ T-A

8 Acce for -7 A-G

8 Donor +1 G-A

9 Acce for -2 A-C

9 Acce for -6 T-G

9 Donor +6 T-A

10 Acce ter -1 G-A

10 Acce for -11 ~-A

10 --. Donor - +3 ~C =

10 Donor +3 i A-G
-11 :Acceptor -8 - ~A _ 11 Donor -6 ~ A-G

11 Donor +~ G-A

11 _ Donor +20 G-C

11 Donor +5 G-T

12 Donor +1 G-A

12 Donor . +2 T-C

~i The position of the oligonucleotides, derived from the reference sequence under GenBank-number U49897, is indicated in columns 1-3. Column 4 specifies the mutation.
C) short deletions Codon-No.Reference sequence Mutated sequence 14 GCAGGAAACTctCTGACTTTGG GCAGGAAACTCTGACTTTGG

38 ATCACTGATCttcTCACTCAAAG ATCACTGATCTCACTCAAAG

54 TTGCGCTTATtTGAG E212 GTCAGT TTGCGCTTATTGAG E212 GTCAGT

69 ATCTAGACCTtctCGTTTAAAGA ATCTAGACCTCGTTTAAAGA

93 TCTGACAAACatcATCAAGATCT TCTGACAAACATCAAGATCT

185 AAAAGAAAACaTGGGGCACAG AAAAGAAAACTGGGGCACAG

210 CACATTTTTCcACTTCTTGAA CACATTTT'TCACTTCTTGAA

220 GCTTCCATGAa ATAACATTCC GCTTCCATGAATAACATTCC

269 AGTACATCAGacat GATCCAAGCC AGTACATCAGGATCCAAGCC

273 CATGGATCCAa cccat tatACCCCCGAACCATGGATC:CAACCCCCGAAC

309 GGCCTTGCCTctct t caCCTGATGAATGGCCTTGGCTCCTGATGAAT

313 CTGGGTGCACcTGATGAATAC CTGGGTGCACTGATGAATAC

352 ATCCTTTGGTgAATTACAG_ ATCCTTTGGTA4TTACAG_ CAAa cttctccccct AGCTGGAGAA

363 GAAGCCAAAGcttCTCCCCCTGG GAAGCCAAAGCTCCCCCTGG

372 GGAGAAGACA cCATCCAAAAT GGAGAAGACACATCCAAAAT

387 CTGTATTACGt GCAGAGAGTT CTGTATTACGGCAGAGAGTT

2a 398 CAAGGAGAAAgtaaG_E11/111 GTGAGGTCAAGGAGAAAG_E11/111 GTGAGGT
GG GG

399 GGAGAAAGTAaG_E111111 GTGAGGTGGGGAGAAAGTAG_E111111 GTGAGGT
GG

401 TTGGTCTTAG_I111E12_gAACTTTGCTGTTGGTCTTAG 1111E12_AACTTTGCT
G

406 GCCACAATACcTCGGCCCTTC GCCACAATACTCGGCCCTTC

407 CACAATACCTcGGCCCTTCTC CACAATACCTGGCCCTTCTC

The deleted nucleotides are presented in small letters.
D)'short insertions Codon-No. Inserted nucleotide gg C

Indels (insertions/deletions) 55 GCTTATTTGAg_E2/I2_gTCAGTACTA
E) larger deletions exon 3 plus flanking sequences 22 Bp, starting at nucleotide 593, codon 198 22 by starting at nucleotide 586, codon 196 265 bp, concerning exons 5-6 exons 1-5 plus flanking sequences exons 9-13 plus flanking sequences 22 by starting at nucleotide 589, codon 197 i1 ' i exons 9-11 plus flanking sequences F) complex rearrangements GA-AC nucleotide 470-471 R157N
Example 2:
DNA-Chip for a neonate screening A DNA-chip is prepared according to the description in example 1. Here as well the 15- to 18-mer oligonucleotide sequences are attached to the carrier by standard techniques. The oligonucleotides are complementary to the respective parts of the reference sequence or the mutated sequences.
The reference sequences are selected from the following genes:
1. Phenylalanine-hydroxylase, PAH (diagnosis of phenylketonuria) 2. Quinoid-dehydropteridin-reductase, QDPR (diagnosis of phenylketonuria) 3. Mitochondrial branched-chain alpha-ketoacid-dehydrogenase, a-peptide, BCKDHA
(diagnosis of maple syrup urine disease).
4. Mitochondrial branched-chain alpha-ketoacid-dehydrogenase, (i-peptide, BCKDHB
(diagnosis of maple syrup urine disease).
5. Branched-chained dihydrolipoamid-transacylase, DBT (diagnosis of maple syrup urine disease).
6. Galactose-1-phosphat uridyltransferase, GALT (diagnosis of galactosaemia).
7. Galactokinase, GALK1 (diagnosis of galactosaemia).
8. UDP-galactose-4-epimerase, GALE (diagnosis of galactosaemia).
9. Cystathion-beta-synthase, CBS (diagnosis of homocystinuria).
10. 5,10-Methylentetrahydrofolat reductase (NADPH), MTHFR (diagnosis of galactosaemia).
11. Methyltetrahydrofolat-L-homocystein-S-methyltransferase, MTR (diagnosis of III

galactosaemia).
12. Biotinidase, BTD (diagnosis of a biotinidase deficiency).
13. Medium chain aryl-CoA-dehydogenase, ACADM (diagnosis of a medium chain acyl-CoA-dehydrogenase-deficiency).
14. Low-density-lipoprotein receptor, LDLR (diagnosis of familiar hypercholesterinaemia) 15. Apolipoprotein B (diagnosis of a familiar defect of apolipoprotein B).
16. "Cystic fibrosis transmembrane conductance regulator", CFTR (diagnosis of a cystic fibrosis).
17. Fibrillin-1, FBN1 (diagnosis of a Marfan-syndrome).
18. "Latent-transforming growth factor", beta-binding protein-2, LTBP2 (diagnosis of a Marian-syndrome).
19. Delta-7-sterolreductase, DHCR7 (diagnosis of a Smith-Lemli-Opitz-syndrome).
20. Sterol 21-hydroxylase, CYP21 (diagnosis of an Adrenogenital syndrome).
21. Sterol 17-hydroxlase, CYP17 (diagnosis of an Adrenogenital syndrome).
The reference sequences of these genes are shown in table 2 and are identified by their respective GenBank code numbers of the National Center for Biotechnology Information (NCBI) are selected.
A selection of oligonucleotides being complementary to functionally characterised mutations is attached to the neonate screening gene chip according to the invention.
These mutations are specified in tables 6.1.1 and 6.12.5 with to their position in the reference sequence and the relevant amino acids.
In total the neonate screening gene chip comprises at least 3.348 hybridising positions for a diagnosis of the 12 most common, treatable inherited diseases. The simultaneous presence of these oligonucleotide sequences on one carrier allows for the simultaneous diagnosis of these hereditary diseases on the basis of one patient probe only. Therefore this DNA-chip is especially suitable for the use in neonatal or prenatal screening programmes.
Table 4 gives further information concerning the diseases to be diagnosed Table 2:

.,.

Gene, Gene symbol Disease GenBank- Hybridising number of the positions ref.-sequence (minimum number) Phenylalaninhydroxylase,Phenylketonuria049897, K03020, 548 PAH

NM 000277, Quinoid Dihydropteridin PhenylketonuriaX04882, M16447 40 Reductase, II

Alpha-ketoaciddehydrogenase,Maple syrupurine M55575 48 mitochondria) a- and disease 214093 ~i-peptide, Dihydrolipoamid-transacylase X66785 Galactose-1-Phosphat GalactosaemiaM60091, L46354- 230 Uridyl-transferase 365, NM 000155, L46691 ~-724 Galactokinase GalactosaemiaM84443, 026401, 4 NM 002044, NM 000154, UDP-Galactose-4-epimeraseGalactosaemiaL41668 16 Cystathion-beta-synthaseHomocystinuriaL14577, 176 X98810-23, X88562, X91910, X87815-6, 5-Methyltetrahydrofolat-homocystein-HomocystinuriaAF025794 4 methyltransferase reductase NM002454 5,10-Methylentetrahydrofolat-Homocystinuria009806, AF105988-32 i,i ', reductase, NADPH 98 Medium chain aeyl-CoA dehy- MCAD M16827, 48 drogenase NM 000016, Biotinidase Biotinidase deficiency 063274, 003274 28 LDL-Receptor Familiar Hyper- L00336-52, 722 cholesterinaemia L29401, Apolipoprotein-B 3500 Familiar defect of Apo-B X04506, M14162 2 Gene Disease GenBank Hybridising psitions number of the ref.-sequences CFTR Cystic Fibrosis M55131, 992 Fibrillin I Marian X63556, L13923,272 LTBP2 Marian 237976 4 Delta-7-SterolreductaseSmith-Lemli-Opitz AF034544 84 CYP21, CYP17 Adrenogenital SyndromeM26856, M3114698 M 13935-6, M 14564, Example 3:
DNA-chip for the diagnosis of a biotinidase deficiency a s In this example the oligonucleotides listed in table 3 are directly attached on a suitable carrier by means of known standard techniques. Analogous to example 1 mostly 15-mers are used, whereas in some cases also 16-mer to 25-mer oligonucleotides can be employed.
The sequences are positioned in order to enable a central position of the deviated sequence relative to the wildtype reference sequence.
Only one single oligonucleotide sequence is attached onto one respective field of the carrier.
In order to diagnose the biotinidase deficiency, 28 functionally characterised mutations are attached onto the chip, resulting in a total of 28 fields. Thereon, e.g. field 1.1 carries the reference sequence section CCAGCATGTCCACTG (table 1, line 1, column 3), which is derived from the gene sequence under GenBank-number U63274, and field 1.2 carries the corresponding mutated sequence CAGTGGATATGCTGG (table 1, line 1, column 4;
mutation underlined).
For the order of the other fields the sequence information of lines 2 to 14 in table 3 are used in the same way.
By selecting suitable primers from the strand complementary to the coding strand of the reference sequence (the "sense-strand") as labels for detecting the hybridisation it is ensured that labelled gene sections are complementary to the oligonucleotides on the chip.
Table 3:
Table 3 contains a fist of nucleic acid sequences, which are attached onto the chip for diagnosing biotinidase deficiency. To keep the table readable, only the central part of the 15- to 25-mer oligonucleotides is shown. The position is specified relatively to the reference sequence by indicating the respective codon number. For each mutated sequence, the native reference sequence shown in the second column is attached at a precisely defined position of the carrier. The sequence parts not shown correspond to the reference sequence.

!i A) Missense- or nonsense-mutations Codon-No. Reference- Mutated sequenceAmino acid-(reference- sequence exchange se uence 128 aTTT GTT Phe-Val 171 GCC ACC Ala-Thr 228 GAT TAT As -T r 323 CAT CGT His-Ar 444 tGAT CAT As -His 451 GGC GAC GI -As 456 CAA CAC Gln-His 489 AAC ACC Asn-Thr 532 ACG ATG Thr-Met 538 CGC TGC Ar -C s B) Splice variants Intron-No. Characteristic Position of Base exchange of Splice site mutation 1 Akze for +56 ~ G-A

The position of the oligonucleotides of the reference sequence is shown in lines 2-4; line 5 specifies the mutated reference sequence; line 1: number of mutation.
C) Small deletions Codon-No. Normal Mutated reference se uence reference se uence 310 TGACAGGAAGtGGCATACACATGACAGGAAGGGCATACACA

D) Indels (insertions/deletions) CTTTTCCTCTgcggctgTTACGTGGTT tcc F) Complex rearrangements 15 by deletion + 11 by ins. in exon D

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Claims (11)

Claims:

1. Nucleotide carrier comprising a selection of oligonucleotide sequences, which are identical or complementary to sections of reference sequences being relevant for at least two genetically caused phenotypes.

2. Nucleotide carrier according to claim 1, characterised in that the selection of oligonucleotides is related to or responsible for a significant share of all variants of a phenotype.

3. Nucleotide carrier according to claim 1 or 2, characterised in reference sequences selected from the group of sequences with the following GenBank numbers:
- K03020, NM 000277, L47726, U49897;
- NM 000320, M16447, X04882;
- Z14093;
- M55575;
- X66785;
- M60091, NM 000155; L46354 to 46365, L46691 to 46724;
- NM 002044, NM 000154, L76927, U26401, M84443;
- L41668;
- NM 000071, L14577, X98810 to X98823, X88562, X87815, X87816, X91910;
- AF025794, NM 002454;
- U09806, AF105988 to AF105998;
- U63274, U03274;
- M16827, M91422 to M91432, NM 000016;
- NM 000527, L00336 to 00352, L29401;
- X04506, M14162;
- NM 000492, M55131;
- NM 000138, X63556, L13923;
- Z37976;
- AF034544;

- M26856, M13935, M13936;
- NM 000102, M14564, M31146.

4. Nucleotide carrier according to one of the claims 1 to 3, characterised in oligonucleotides being selected from the group of the sequences specified in tables 6.1.1 to 6.12.5.

5. Nucleotide carrier according to one of the claims 1 to 4, characterised in oligonucleotides having a length of 16 to 25 nucleotides, preferably having a length of 15 to 18 nucleotides.

6. Nucleotide carrier according to one of the claims 1 to 5, characterised in a carrier consisting of gold-coated glass.

7. Nucleotide carrier according to one of the above mentioned claims, characterised in a combination of oligonucleotides for the detection of phenylketonuria and galactosaemia.

8. Nucleotide carrier according to claim 7, characterised in oligonucleotides for the detection of an biotinidase deficiency.

9. Use of a nucleotide carrier according to one of claims 1 to 6 for the simultaneous diagnosis of at least two diseases selected from the following group:
phenylketonuria;
maple syrup urine disease;
galactosaemia;
homocystinuria;
biotinidase deficiency;
medium chain acyl-CoA dehydrogenase deficiency;
familiar hypercholesterinaemia;
familiar defect of apolipoprotein-B;

cystic fibrosis;
Marfan-syndrome;
Smith-Lemli-Opitz syndrome;
adrenogenital syndrome.

10. Use of a nucleotide carrier according to one of the claims 1 to 8 for a neonate screening.

11. Use of a nucleotide carrier according to one of claims 1 to 8 for a prenatal screening