CN118159667A - Method for detecting repeated spreading sequences - Google Patents

Abstract

The present disclosure relates, inter alia, to a method of detecting the presence or absence of a repeat extension sequence in two or more genes in a nucleic acid sample obtained from a subject, the method comprising: i) Contacting a nucleic acid sample of each of the two or more genes with: a) A gene-specific primer that binds to a different target sequence of each gene, wherein the genes comprise nucleotide repeats, and wherein the different target sequences are upstream or downstream of the nucleotide repeats, and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeats and on the opposite strand bound by the gene-specific primer; and ii) analyzing the amplification product.

Description

Method for detecting repeated extended sequences

The present disclosure relates to the field of molecular biology. In particular, the specification teaches methods for detecting the presence or absence of repeat expansion sequences in two or more genes in a nucleic acid sample, and methods for screening for multiple repeat expansion diseases in a subject.

Background Art

Expansions of simple sequence repeats scattered throughout the human genome are now known to directly cause more than 35 human diseases. The most common repeat expansion diseases are caused by trinucleotide repeats, although tetranucleotide, pentanucleotide, hexanucleotide, and even dodecanucleotide repeat expansions have also been identified as potential mutations in other diseases.

Repeat expansion diseases often present with distinct phenotypes and are often difficult to distinguish by signs and symptoms alone due to extensive clinical overlap and other concomitant phenotypes. Therefore, molecular genetic testing is necessary to identify pathogenic mutations to confirm the disease state in symptomatic individuals. Several diseases are caused by common repeat expansion mutations located at different loci, such as CGG or GCG repeat expansions causing fragile X syndrome (FXS), several types of distal oculopharyngeal myopathy, oculopharyngeal muscular dystrophy, and developmental epileptic encephalopathy, CCG repeat expansions causing a type of intellectual development and oculopharyngeal myopathy with leukoencephalopathy, CAG repeat expansions causing Huntington's disease and several types of spinocerebellar ataxia (SCA), CTG repeat expansions causing myotonic dystrophy type 1, Huntington's disease-like 2, and Fuchs corneal endothelial dystrophy, and TTTCA pentanucleotide repeat expansions causing several types of familial adult myoclonic epilepsy and spinocerebellar ataxia. Multiple rounds of genetic testing may be required to achieve a correct diagnosis for an affected individual, resulting in additional cost and time.

Therefore, there exists a need to overcome or at least alleviate one or more of the above mentioned problems.

Summary of the invention

Disclosed herein is a method for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject, the method comprising: i) contacting the nucleic acid sample under amplification conditions for each of the two or more genes with: a) gene-specific primers that specifically bind to different target sequences of each gene, wherein the genes comprise nucleotide repeat sequences, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequences, and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primers; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; and ii) analyzing the amplification products.

Disclosed herein is a kit for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject, the kit comprising: a) gene-specific primers that specifically bind to different target sequences of each of the two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene.

Disclosed herein is a composition comprising a nucleic acid sample obtained from a subject, the composition comprising a) gene-specific primers that specifically bind to different target sequences of each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer of each gene;

Gene-specific primers and universal primers can generate one or more amplification products from each gene.

Disclosed herein is a method for screening for one or more multiple repeat expansion diseases in a subject, the method comprising: i) contacting a nucleic acid sample from the subject with: a) gene-specific primers that specifically bind to different target sequences in each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; and ii) analyzing the amplification products to screen for one or more multiple repeat expansion diseases in the subject.

Disclosed herein is a method for screening for one or more multiple repeat expansion diseases in a subject and treating the subject, the method comprising: i) contacting a nucleic acid sample from the subject with: a) gene-specific primers that specifically bind to different target sequences in each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; ii) analyzing the amplification products to screen for one or more multiple repeat expansion diseases in the subject; and iii) treating the subject found to have at least one multiple repeat expansion disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the present invention will now be described by way of non-limiting examples only, with reference to the accompanying drawings, in which:

Fig. 1. schematic diagram of FMR1 and AFF2 dual TP-PCR reaction.Indicated locus specific flanking primer annealing position (black and gray arrows, wherein its end is marked with a star symbol), as is the TP primer annealing position in the repetition (black and gray arrows).Gray arrow indicates poor amplification, because the TP primer is farther from the locus specific primer, or because of the mispairing of the mediation of interruption.Shown the expected electrophoretogram pattern of the sample carrying only normal FMR1 and normal AFF2 allele (A), extended FMR1 allele and extended AFF2 allele (C).Black rectangle represents FMR1 CGG or AFF2CCG trinucleotide.Gray rectangle represents non-CGG or non-CCG trinucleotide as shown above each rectangle.

Fig. 2. electrophoretogram of FMR1 (FAM/dark grey) and AFF2 (HEX/light grey) dual TP-PCR products from normal, FMR1 premutation and FMR1 full mutation male and female DNA samples. Electrophoretogram shows both FAM and HEX (left), only FAM (middle) and only HEX (right) fluorescence channels. Dark grey peak indicates FAM-labeled FMR1 TP-PCR product, while light grey peak indicates HEX-labeled AFF2 TP-PCR product. The threshold repeat size separating normal allele from extended allele is indicated by vertical dotted line.

Fig. 3. Electrophoretogram of FMR1 (FAM/dark gray) and AFF2 (HEX/light gray) dual TP-PCR products from AFF2 premutation and full mutation DNA samples. Electrophoretograms show both FAM and HEX (left), FAM only (middle) and HEX only (right) fluorescence channels. Dark gray peak indicates FAM-labeled FMR1 TP-PCR product, while light gray peak indicates HEX-labeled AFF2 TP-PCR product. The threshold repeat size separating normal alleles from extended alleles is indicated by vertical dotted lines.

Figure 4. AFF2 CCG repeat size and structure distribution. A, Population distribution of AFF2 CCG repeat size (x-axis) and frequency (y-axis) in African American (gray fill), Caucasian (unfilled), Chinese (black fill), Indian (dark gray thick stripes), and Malay (gray forward slashes) populations. B, Comparison of allele frequency distribution in Chinese in Zhong et al. (gray) and this study (black). C, Comparison of allele frequency distribution in Caucasians in Zhong et al. (light gray) and this study (gray). D, Depicts the population distribution of X chromosomes with different FMR1 CGG and AFF2CCG repeat size combinations (top), and heat maps of population repeat size distribution and abundance (bottom) of common and variant AFF2 alleles.

Fig. 5 . spectrum of AFF2 CCG repeat structure and corresponding TP-PCR electrophoretogram pattern.A, common normal allele with rs868914124 (T) reference nucleotide.B, variant normal allele with rs868914124 (C) variant nucleotide.C, variant normal allele with rs868914124 (T) reference nucleotide and rs1389911365 (T) variant nucleotide.D, full mutation allele with rs868914124 (C) variant nucleotide and three consecutive non-CCG interruptions. "+" indicates non-CCG interruption, while "+++" indicates three consecutive non-CCG interruptions. The isolated peak of 111bp on the left is produced by annealing of TP primers upstream of the repeat segment. The TP-PCR product produced by the first repeat of the common allele of rs868914124 (T) migrates to a 138bp fragment. The TP-PCR product generated from the first repeat of the rs868914124 (C) rare allele migrated as a 132 bp fragment. Interruptions within the repeat resulted in mismatched TP primer pairing, which resulted in peak-free gaps within the peak cluster.

Figure 6. (A) AFF2CCG repeat structure and corresponding TP-PCR electrophoresis pattern of full mutation male FX0229 and (B) his full mutation uncle FX0230. A, full mutation allele carries rs868914124 (C) rare variant and three consecutive non-CCG interruptions at repeat 8-10. B, full mutation allele with rs868914124 (C) rare variant and multiple non-CCG interruptions. "+++" indicates three consecutive non-CCG interruptions.

Fig. 7. (A) AFF2 CCG repeat structure and corresponding TP-PCR electrophoresis pattern of full mutation male DNA_25926 and (B) its premutation progeny DNA_3802. A, full mutation allele carrying rs868914124 (C) rare variant and three consecutive non-CCG interruptions at repeat 6-8. B, normal allele carrying 22 CCG repeats of rs868914124 (T) common reference nucleotides, and premutation allele with about 137 repeats of rs86891412 (C) rare variant and three consecutive non-CCG interruptions at repeat 6-8. "+++" indicates three consecutive non-CCG interruptions.

Figure 8. Multiplex three-primer PCR of seven common SCA repeat loci. Schematic diagram of SCA1, SCA2, SCA3, SCA6, SCA7, SCA12 and DRPLA repeat structure and primer annealing positions (A) and expected TP-PCR electropherograms for each repeat locus (B). Asterisks indicate fluorophore labels. The upper limit of normal allele repeat size for each locus is indicated by vertical dashed lines.

Fig. 9. seven TP-PCR capillary electropherograms are generated from DNA samples that are negative for expansion at any one of the seven SCA repeat loci. The upper figure shows the electrophoretic peaks of TP-PCR products from all seven repeat loci visible when all four fluorophore channels (Fam, Vic, Ned and Pet) are turned on. The following four figures show the same capillary electropherograms when one fluorophore channel is turned on at a time. The upper limit of the normal allele repeat size of each locus is indicated by a vertical dotted line.

Figure 10. Seven-fold TP-PCR capillary electropherogram results of DNA samples affected by SCA. For each sample, only the electropherogram in which the fluorophore channel shows the repeat expansion at the relevant SCA locus is shown. The samples are positive for CAG repeat expansion in ATXN1 (top row), ATXN2 (second row), ATXN3 (third row), CACNA1A (fourth row), ATXN7 (fifth row), PPP2R2B (sixth row) and ATN1 (bottom row). The upper limit of the normal allele repeat size for each locus is indicated by the vertical dotted line.

DETAILED DESCRIPTION

The present specification teaches a method for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject, the method comprising: i) contacting the nucleic acid sample under amplification conditions for each of the two or more genes with: a) gene-specific primers that specifically bind to different target sequences of each gene, wherein the genes comprise nucleotide repeat sequences, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequences, and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primers; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; and ii) analyzing the amplification products.

In one embodiment, the method is a simultaneous method for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject.

Without being bound by theory, the inventors have developed a strategy for the presence of repeat expansion mutations in two or more suspicious genes of a patient, and described its application in the present disclosure using two examples. The strategy uses a single tube assay to screen multiple genetic loci that cause different repeat expansion diseases, which are caused by the expansion of the same type of repeats. The specific assay shown herein uses multiple three-primer PCR (TP-PCR) to detect expansion mutations involving, for example, trinucleotide repeat sequences present in different genes by using differentially labeled locus-specific flanking primers and universal three-primer (TP) primers. Expansion products at any locus that show repeat sizes within the pathogenic size range, or that exceed the maximum normal repeat size, can be quickly identified and sized by capillary electrophoresis. A single amplification reaction provides a reliable one-step mutation screening of multiple disease genes, thereby greatly shortening the long process of diagnosis of affected individuals. The strategy can be applied to simultaneously screen any group of disease genes that share the same repeat sequence.

In one embodiment, the amplified products are separated according to size. In one embodiment, a change in the size of the amplified products of a gene compared to a reference indicates the presence of a repeat expansion sequence in the gene. In one embodiment, no change in the size of the amplified products of a gene compared to a reference indicates the absence of a repeat expansion sequence in the gene.

The terms "detect," "determine," "measure," "evaluate," "assess," and "determine" are used interchangeably herein to refer to any form of measurement, including determining the presence or absence of an element. These terms include quantitative and/or qualitative determination. Assessments may be relative or absolute.

The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

As used herein, the term "nucleic acid" and equivalent terms, such as "polynucleotide" refer to nucleotides of any length in polymeric form, such as ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNA), which contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. Nucleic acids can be double-stranded or single-stranded. Reference to single-stranded nucleic acids includes reference to sense or antisense strands. The backbone of a polynucleotide may include sugar and phosphate groups as commonly found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include complements, fragments and variants of nucleosides, nucleotides, deoxynucleosides and deoxynucleotides or their analogs.

The term "primer" is used herein to refer to any single-stranded oligonucleotide sequence that can be used as a primer in, for example, PCR technology. Therefore, "primer" according to the present disclosure refers to a single-stranded oligonucleotide sequence that can serve as a starting point for the synthesis of a primer extension product that is substantially identical to the nucleic acid strand to be replicated (for a forward primer) or substantially identical to the reverse complement of the nucleic acid strand to be replicated (for a reverse primer). Primers may be suitable for, for example, PCR technology. Single strands include, for example, hairpin structures formed by single-stranded nucleotide sequences.

The design of the primer, such as its length and specific sequence, depends on the nature of the target nucleotide sequence and the conditions under which the primer is used, such as temperature and ionic strength.

Primer can be made up of nucleotide sequence as described herein, or can be 10,15,20,25,30,35,40,45,50,75,100 or more nucleotides that comprise sequence as described herein or fall within sequence as described herein, condition is that they are suitable for specifically binding target sequence under stringent conditions.In one embodiment, the length of primer sequence is less than 35 nucleotides, for example the length of primer sequence is less than 34,33,32,31,30,29,28,27,26,25,24,23,22 or 21 nucleotides.

The length or sequence of the primer or probe may be slightly modified to maintain the specificity and sensitivity required in a given situation. In one embodiment of the present disclosure, the length of the probe and/or primer described herein may be, for example, extended by 1, 2, 3, 4, or 5 nucleotides in either direction, or reduced by 1, 2, 3, 4, or 5 nucleotides.

Primer sequences can be synthesized using any method known in the art.

The term "complementary" refers to base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are considered complementary when the nucleotides of one strand, after optimal alignment and comparison, and with appropriate nucleotide insertions or deletions, are paired with at least about 80% of the nucleotides of the other strand (usually at least about 90% to 95% of the nucleotides of the other strand, and more preferably about 98% to 100% of the nucleotides of the other strand). Alternatively, complementarity exists when an RNA or DNA strand will hybridize with its complement under selective hybridization conditions. Typically, selective hybridization will occur when there is at least about 65% complementarity, preferably at least about 75%, and more preferably at least about 90% complementarity over a stretch of at least 14 to 25 nucleotides.

As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide. The resulting (usually) double-stranded polynucleotide is a "hybrid." The proportion of a population of polynucleotides that form stable hybrids is referred to herein as the "degree of hybridization."

Typically, hybridization conditions will include a salt concentration of less than about 1M, more usually less than about 500mM and less than about 200mM. The hybridization temperature can be as low as 5°C, but is usually greater than 22°C, more usually greater than about 30°C, and preferably greater than about 37°C. Hybridization is usually carried out under stringent conditions (i.e., conditions under which the probe will hybridize with its target subsequence). Stringent conditions are sequence-dependent and are different under different environments. Longer fragments may require higher hybridization temperatures for specific hybridization. Since other factors may affect the stringency of hybridization, including the base composition and length of the complementary chain, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Typically, stringent conditions are selected to be about 5°C lower than the thermodynamic melting point (Tm) of a specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the probes complementary to the target sequence hybridize with the target sequence in a state of equilibrium (under defined ionic strength, pH, and nucleic acid composition). Typically, stringent conditions include a salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH of 7.0 to 8.3 and a temperature of at least 25° C. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridization.

"Specific binding" or "specific hybridization" of a primer to a target sequence means that under the experimental conditions used, for example, under stringent hybridization conditions, the primer forms a duplex (double-stranded nucleotide sequence) with part of the target sequence region or with the entire target sequence (as required), and under these conditions, the primer does not form a duplex with other regions of the nucleotide sequence present in the sample to be analyzed.

The term "repeat expansion sequence" may refer to a nucleotide repeat sequence that has undergone an expansion mutation resulting in the nucleotide repeat sequence being elongated beyond the normal repeat size.

In one embodiment, the repeat expansion sequence is a trinucleotide repeat expansion sequence. In other embodiments, the repeat expansion sequence is a tetranucleotide or pentanucleotide repeat expansion sequence or a repeat expansion sequence of other lengths, such as a repeat sequence of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

In one embodiment, the nucleotide repeat sequence is a trinucleotide repeat sequence. In one embodiment, the trinucleotide repeat sequence is selected from (CGG) _n , (CCG) _n , (CAG) _n , (CTG) _n , (GCC) _n , (GGC) _n , (GAA) _n or (TTC) _n , wherein n is 2 to 200 or greater. (CGG) _n repeat sequences include (GCG) _n and (GGC) _n . Similarly, (CCG) _n sequences include (CGC) _n and (GCC) _n , and (CTG) _n sequences include (TGC) _n and (GCT) _n .

A "universal primer" as referred to herein can bind to a "common target sequence" shared by two or more genes, wherein the "common target sequence" is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer.

A "gene-specific primer" as mentioned herein may refer to a primer that specifically binds to a particular gene but does not bind to other genes.

The term "gene" is widely used to refer to any nucleic acid associated with a biological function. A gene generally includes a coding sequence and/or a regulatory sequence required for the expression of such a coding sequence. The term gene can be applied to a specific genomic sequence, as well as to a cDNA or mRNA encoded by the genomic sequence. A gene also includes a non-expressed nucleic acid segment, which, for example, forms a recognition sequence for other proteins. Non-expressed regulatory sequences include promoters and enhancers, to which regulatory proteins (e.g., transcription factors) bind, resulting in transcription of adjacent or nearby sequences.

In one embodiment, the universal primer binds to a common target sequence comprising or consisting of 5, 6, 7, 8, 9, 10 or more consecutive trinucleotide repeats. In one embodiment, the universal primer binds to (CGG) ₅ (SEQ ID NO: 15). In one embodiment, the universal primer binds to (CTG) ₅ (SEQ ID NO: 16).

In one embodiment, the universal primer binds to a common target sequence that comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive repeating units of tetranucleotides, pentanucleotides or more. In one embodiment, the pentanucleotide repeat sequence is (TTTCA) _n , which includes (CATTT) _n and (ATTTC) _n .

In one embodiment, the universal primer comprises a unique tail sequence. In one embodiment, the universal primer comprises a unique 5' tail sequence. The term "unique tail sequence" or "unique 5' tail sequence" refers to a sequence that does not hybridize to any region in any gene or intergenic region in a nucleic acid sample under stringent conditions, and is used to detect the presence or absence of a repeat expansion sequence.

In one embodiment, the method includes providing a tail primer that specifically binds to a unique 5' tail sequence of a universal primer. Adding a unique 5' tail sequence to a universal primer and providing a tail primer that specifically binds to the unique 5' tail sequence can improve the accuracy of repeat size determination.

In one embodiment, the gene-specific primer is labeled. Each gene-specific primer can be labeled, for example, with a different fluorophore so that the amplified products from each gene can be distinguished from each other. For example, FAM or Hex labels can be used.

In one embodiment, the fluorescent label can be active in the blue, yellow, green and far red regions of the spectrum. In a preferred embodiment, non-limiting examples of fluorescent labels that can be used for the methods of the present disclosure include: fluorescent labels or reporter dyes, such as 6-carboxyfluorescein (6FAM ^™ ), NED ^™ (Applera Corporation), HEX ^™ or VIC ^™ (Applied Biosystems); TAMRA ^™ markers (Applied Biosystems, CA, USA), ROX. It will be appreciated by those skilled in the art that other alternative fluorescent labels may also be used in the methods according to the present disclosure.

In another embodiment, chemiluminescent labels, such as ruthenium probes, and radioactive labels, such as tritium in the form of tritiated thymidine, can be used. 32-Phosphorus can also be used as a radioactive label.

Alternatively, the label may be selected from electroluminescent tags, magnetic tags, affinity or binding tags, nucleotide sequence tags, position-specific tags and/or tags with specific physical properties (e.g. different size, mass, rotation, ionic strength, dielectric properties, polarization or impedance).

In one embodiment, the detectable label can be attached directly or indirectly to the primer. In one embodiment, the labeled primer is a reverse primer. In one embodiment, the detectable label comprises a fluorescent moiety attached to the 5' end of the probe. In a most preferred embodiment, the label is selected from 6-FAM and NED.

In an alternative embodiment, nucleic acids are detected with nucleic acid intercalating fluorophores. Preferably, the intercalating fluorophore is SYBRGreen or EvaGreen, etc. It will be appreciated by those skilled in the art that other intercalating fluorophores active in the blue, yellow, green, and far-red regions of the spectrum may be used. It will be further appreciated that other intercalating fluorophores may be used in accordance with the present disclosure.

The term "sample" mentioned herein may be derived from a biological fluid, cell, tissue, organ or organism, including a nucleic acid or a mixture of nucleic acids with at least one nucleic acid sequence for screening of copy number variation. In certain embodiments, the sample has at least one nucleic acid sequence, and it is suspected that its copy number has been changed. Such samples include but are not limited to sputum/oral fluid, amniotic fluid, blood, blood fractions or fine needle biopsy samples, urine, peritoneal fluid, pleural fluid, etc. Although samples are usually taken from human subjects (such as patients), samples can be taken from any mammal, including but not limited to dogs, cats, horses, goats, sheep, cattle, pigs, etc. Samples can be used directly or after pretreatment of the characteristics of the sample as obtained from biological sources. For example, this pretreatment can include preparing plasma from blood, diluting viscous fluids, etc. The method of pretreatment may also be related to but not limited to filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, freeze-drying, concentration, amplification, nucleic acid fragmentation, inactivation of interfering components, addition of reagents, cracking, etc. Where such pretreatment methods are employed on a sample, such pretreatment methods typically result in retention of the nucleic acid of interest in the test sample, sometimes at a concentration proportional to that in an untreated test sample (e.g., i.e., a sample that has not been subjected to any such pretreatment methods).

A control sample can be a negative or positive control sample. A "negative control sample" or "unaffected sample" refers to a sample that includes a nucleic acid known or expected to have a repetitive sequence with a number of repeats within the non-pathogenic range. A "positive control sample" or "affected sample" is known or expected to have a repetitive sequence with a number of repeats within the pathogenic range. The repetition of the repetitive sequence in the negative control sample is generally not extended beyond the normal range, while the repetition of the repetitive sequence in the positive control sample is generally extended beyond the normal range. Therefore, the nucleic acid in the test sample can be compared to one or more control samples.

The term "biological fluid" herein refers to a liquid taken from a biological source, and includes, for example, blood, serum, plasma, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, saliva, etc. As used herein, the terms "blood", "plasma" and "serum" explicitly encompass fractions or processed portions thereof. Similarly, in the case where the sample is taken from a biopsy, swab, smear, etc., the "sample" explicitly encompasses processed fractions or portions derived from a biopsy, swab, smear, etc.

"Label" refers to a reporter molecule (eg, a fluorophore) that is capable of producing a measurable signal and is covalently or non-covalently attached to a polynucleotide.

In one embodiment, the method includes using size separation technology or sequencing technology to analyze the amplified product. Size separation technology can be a technology based on electrophoresis (such as capillary electrophoresis). For example, capillary electrophoresis performed by size separation polymer can be used. Fluorescently labeled PCR products (via primers using 5' end labels) are detected via laser excitation when they migrate and resolve through polymer-filled capillaries. Another size separation technology is slab gel PAGE (polyacrylamide gel electrophoresis). PCR products can be fluorescently labeled using 5' end-labeled primers, and detected via laser excitation when they migrate and resolve through slab gels. PCR products can also be radiolabeled via 5' end-labeled primers or via incorporation of radioisotope-labeled nucleotides during the PCR process, and detected by exposing the slab gel to X-ray film. Sequencing technology can be next generation sequencing. By using long read next generation sequencing, PCR products can be sequenced, and all reads belonging to the target gene can be arranged from the shortest to the longest, or vice versa.

As mentioned herein, two or more genes can be adjacent to each other or close to each other on the same chromosome. Alternatively, they can be on different chromosomes.

In one embodiment, the two or more genes consist of or include FMR1 and AFF2. FMR1 and AFF2 are located on the same chromosome (X) on the long arm in adjacent chromosome bands. This provides a simple method for simultaneously testing two or more genes including FMR1 and AFF2, either of which can cause the disease exhibited by the patient.

In one embodiment, the method comprises contacting a nucleic acid sample with: a) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 1; b) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 2; and c) a universal primer comprising or consisting of a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 3 or (CGG) ₅ (SEQ ID NO 15).

In one embodiment, the two or more genes include or are selected from SCA1, SCA2, SCA3, SCA6, SCA7, SCA12 and DRPLA. In one embodiment, the two or more genes consist of two, three, four, five, six or seven or more genes, and the genes include or are selected from SCA1, SCA2, SCA3, SCA6, SCA7, SCA12 and DRPLA.

In one embodiment, the two or more genes are HTT and JPH3.

In one embodiment, the two or more genes consist of ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, and ATN1.

In one embodiment, the two or more genes include or are selected from ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, ATN1, TBP, ATXN8, ATXN8OS, AR, HTT, JPH3, TCF4 and DMPK, and consist of two, three, four, five, six, seven, eight, nine, ten or more or all of these genes.

In one embodiment, the two or more genes include or are selected from FMR1, AFF2, NUTM2BAS1, LRP12, GIPC1, NOTCH2NLC, RILPL1, PABPN1 and ARX, and consist of two, three, four, five, six, seven, eight or all of these genes.

In one embodiment, the two or more genes include or are selected from DAB1, SAMD12, STARD7, TNRC6A and RAPGEF2, and consist of two, three, four or all of these genes.

In one embodiment, the method comprises contacting the nucleic acid sample with: a) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:7; b) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:8; c) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9; d) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:10; e) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:11; f) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:12; and/or g) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:13. NO:13, and h) a universal primer comprising or consisting of a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:14 or (CTG) ₅ (SEQ ID NO:16).

Disclosed herein is a kit for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject, the kit comprising: a) gene-specific primers that specifically bind to different target sequences of each of the two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene.

Disclosed herein is a composition comprising a nucleic acid sample obtained from a subject, the composition comprising a) gene-specific primers that specifically bind to different target sequences of each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene.

Disclosed herein is a method for screening for one or more multiple repeat expansion diseases in a subject, the method comprising: i) contacting a nucleic acid sample from the subject with: a) gene-specific primers that specifically bind to different target sequences in each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; and ii) analyzing the amplification products to screen for one or more multiple repeat expansion diseases in the subject.

The term "multiple repeat expansion disease" refers to a genetic disorder caused by an increase in the number of DNA repeat motifs (e.g., trinucleotide repeat motifs) in certain genes above a normal, stable threshold (the threshold is different for each gene). The term is intended to include all disorders of this nature and includes the disorders listed in Table 1.

Table 1. Diseases caused by expansions of different repeat motifs

In one embodiment, the one or more multiple repeat expansion diseases include or consist of one or more diseases listed in Table 1.

In one embodiment, the one or more multiple repeat expansion diseases comprise or consist of a disease comprising or selected from the group consisting of Fragile X syndrome (FXS), Fragile X-associated primary ovarian insufficiency (FXPOI), Fragile X-associated tremor/ataxia syndrome (FXTAS), and Fragile XE non-syndromic intellectual disability (FRAXE NSID).

In one embodiment, the method distinguishes between FXS, FXPOI, FXTAS, and FRAXE NSID.

In one embodiment, the one or more multiple repeat expansion diseases are spinocerebellar ataxia (SCA) and/or dentatorubral pallidum atrophy with Lewy bodies (DRPLA).

In one embodiment, the method distinguishes between SCA and DRPLA. In one embodiment, the method distinguishes between different SCA types and DRPLA.

In one embodiment, the one or more multiple repeat expansion diseases are Huntington's disease (HD) and Huntington's disease-like 2 (HDL2).

In one embodiment, the method distinguishes between Huntington's disease (HD) and Huntington's disease-like 2 (HDL2).

In one embodiment, the method distinguishes between different types of oculopharyngeal distal myopathy (OPDM), oculopharyngeal muscular dystrophy (OPMD), oculopharyngeal myopathy with leukoencephalopathy (OPML), and developmental epileptic encephalopathy (DEE).

In one embodiment, the method distinguishes between different types of familial adult myoclonic epilepsy (FAME) and spinocerebellar ataxia (SCA)37.

"Subject" or "patient" refers to any individual subject in need of treatment, including humans, cows, horses, pigs, goats, sheep, dogs, cats, guinea pigs, rabbits, chickens, insects, etc. Subjects are also intended to include any subject participating in a clinical research trial but not showing any clinical signs of disease or a subject participating in an epidemiological study, or a subject used as a control.

Disclosed herein is a method for screening for one or more multiple repeat expansion diseases in a subject and treating the subject, the method comprising: i) contacting a nucleic acid sample from the subject with: a) gene-specific primers that specifically bind to different target sequences in each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; ii) analyzing the amplification products to screen for one or more multiple repeat expansion diseases in the subject; and iii) treating the subject found to have at least one multiple repeat expansion disease.

The terms "treat", "treatment", and the like also include alleviating, reducing, alleviating, improving, or otherwise inhibiting the effects of a condition for at least a period of time. It should also be understood that the terms "treat", "treatment", and the like do not mean that a condition or its symptoms are permanently alleviated, reduced, alleviated, improved, or otherwise inhibited, and thus also encompass temporary alleviation, reduction, alleviation, improvement, or other inhibition of a condition or its symptoms.

The method of treating a subject may include administering a drug to the subject, or may include providing early intervention management to the subject.

The term "administering" means bringing an inhibitor as referred to herein into contact with a subject, applying it, injecting it, infusing it or providing it to a subject.

Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to mean the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used in this specification, the singular forms "a", "an", and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a single method as well as two or more methods; reference to "an agent" includes a single agent as well as two or more agents; reference to "the disclosure" includes single and multiple aspects of the disclosure teachings; and so forth. Aspects taught and enabled herein are encompassed within the term "invention". Any variations and derivatives are contemplated.

Reference in this specification to any prior publication (or information derived therefrom), or reference to any known matter, is not and should not be taken as an acknowledgement or acknowledgment or any form of opinion that the prior publication (or information derived therefrom) or known matter constitutes part of the common general knowledge in the field to which this specification relates.

Example

Certain embodiments of the present invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the scope of the subject matter described above.

Example 1: FRAXA/FRAXE

Materials and methods

Biological samples

Genomic DNA was extracted from 408 unrelated and anonymous umbilical cord bloods of 161 Chinese male infants, 158 Malay male infants and 89 Indian male infants born in National University Hospital, Singapore. Another 44 Caucasian male DNA samples and 14 African American male DNA samples from human variant DNA groups (HD100CAU and HD100AA-2) were purchased from Coriell Cell Repositories (Camden, New Jersey, USA). The archived and previously characterized genomic DNA consisting of 40 normal, 17 FMR1 premutation positive, 23 FMR1 full mutation positive and 6 AFF2 expansion positive samples was used in the determination validation. The normal and FMR1 CGG repeat expansion positive samples of de-identification were obtained from KK Women'sand Children's Hospital. The AFF2 CCG repeat expansion positive samples were de-identified archived samples from Baylor College of Medicine (Houston, TX, USA) and The University of Adelaide (Adelaide, South Australia, Australia). Four of the six AFF2 CCG repeat expansion positive samples were related. FX0230 and FX0229 were uncle and nephew, respectively, while DNA_25926 and DNA_3802 were father and daughter.

Duplex TP-PCR of FMR1 and AFF2 trinucleotide repeats

Dual screening for AFF2 and FMR1 triplet repeat expansions utilized a three-primer (TP) PCR approach involving four primers, Fam-tagged FMR1-R (5'-AGCCCCGCACTTCCACCACCAGCTCCTCCA-3' (SEQ ID NO: 1)), Hex-tagged AFF2-F (5'-CCATGTCGCGGCTTCTAGCTGTCCAGGCTCC-3' (SEQ ID NO: 2)), and shared primers TP (5'-TGCTCTGGACCCTGAAGTGTGCCGTTGATACGGCGGCGGCGGCGG-3' (SEQ ID NO: 3)) and tail (5'-TGCTCTGGACCCTGAAGTGTGCCGTTGATA-3' (SEQ ID NO: 4)). Each 15 μl PCR reaction contained 100 ng of genomic DNA, 2.5× Q-Solution (Qiagen, Hilden, Germany), 1× PCR buffer (Qiagen) containing 1.5 mM MgCl ₂ , 2 mM dNTP (dGTP/dCTP to dATP/dTTP ratio of 5:1) (Roche Applied Science, Mannheim, Germany), 0.6 μM each of AFF2-F, FMR1-R and tail primers, 0.0006 μM TP primer and 5 U of HotStarTaq DNA polymerase (Qiagen). An initial 15-minute enzyme activation was performed at 95° C., followed by 40 cycles of 99° C. 45 seconds, 55° C. 45 seconds and 70° C. 8 minutes (with 15-second increments for each extension cycle), and a final extension at 72° C. for 10 minutes.

Standard PCR spanning the FMR1 and AFF2 trinucleotide repeats

Standard/conventional PCR across the FMR1 CGG repeat utilized primers 5'-F1 and Fam-tagged FMR1-R at 0.6 μM each, while PCR across the AFF2 CCG repeat utilized Hex-tagged AFF2-F and AFF2-R (5'-CGCTGCGGGCTCAGGCGGGCT-3' (SEQ ID NO:5)). PCR reactions and cycling conditions were similar to the above-mentioned duplex TP-PCR except that 50 ng of genomic DNA was used in each reaction.

Capillary electrophoresis

The aliquot of 2 μ l duplex TP-PCR product is mixed with 9 μ l Hi-Di ^TM formamide and 0.5 μ l GeneScan ^TM 500ROX ^TM dye size standard (Applied Biosystems, Foster City, California, USA), denatured at 95 ℃ for 5 minutes, cooled to 4 ℃, and resolved in the 3130xl genetic analyzer (Applied Biosystems) using the 36 cm capillary filled with POP-7 ^TM polymer. The mixture is injected electrically for 5 seconds at 1 kV, and electrophoresed for 40 minutes at 60 ℃. GeneMapper ^TM software v4.0 (Applied Biosystems) is used to analyze. If the extended allele is detected after the initial capillary electrophoresis (CE) runs, then use an electric injection of 5 seconds at 10 kV, and electrophoresis for 40 minutes at 60 ℃ to run the second CE.

An aliquot of 1 μl of the 20th diluted standard PCR product was mixed with 9 μl of Hi-Di ^TM formamide (Applied Biosystems) and 0.3 μl of GeneScan ^TM 500ROX ^TM dye size standard (Applied Biosystems), denatured at 95°C for 5 minutes, cooled to 4°C, and resolved in a 3130xl genetic analyzer (Applied Biosystems) using a 36 cm capillary filled with POP-7 ^TM polymer. The mixture was electrokinetically injected at 1.2 kV for 23 seconds and electrophoresed at 60°C for 20 minutes.

Post-CE analysis was performed using GeneMapper ^™ software v4.0 (Applied Biosystems).

Data interpretation

Primer AFF2-F anneals to the AFF2 sequence immediately upstream of the CCG repeats of the AFF2 sequence and produces Hex-labeled TP-PCR amplicons, while primer FMRI-R anneals to the FMR1 sequence immediately downstream of the CGG repeats of the FMR1 sequence and produces Fam-labeled TP-PCR products. Both TP-PCR reactions use common tri-primer (TP) primers and tail primers, and their products can be analyzed separately or together using different fluorescence detection channels. The TP primers are designed to anneal optimally to any stretch of five CCG trinucleotides on the sense strand of AFF2 or the antisense strand of FMR1. When there is a continuous uninterrupted stretch of more than five repeats, the electropherogram will show a series of continuous peaks that are 3 bp apart from each other. If there is a non-CCG interruption, the electropherogram will show a gap of about 18 bp between the fluorescent peaks, which is equivalent to the absence of 5 fluorescent peaks (Figure 1). The repeat size of an uninterrupted stretch is the total number of consecutive fluorescence peaks plus four (the first fluorescence peak is generated by the TP primer annealing to the first 5 repeats). For alleles with non-CCG interruptions, the repeat size is the total number of consecutive fluorescence peaks plus the total number of deletion peaks, plus four. In heterozygous females, a decrease in fluorescence intensity will be seen for peaks above a certain size; the repeat size of the smaller allele can be derived from the number of peaks before the decrease, while the repeat size of the larger allele can be derived from the total number of peaks (data not shown). For the AFF2 TP-PCR reaction, the leftmost isolated peak is not generated within the repeat sequence, but is generated by the annealing of the TP primer to the (CCG) ₄ CCT (SEQ ID NO: 6) sequence immediately upstream of the CCG repeat, which is defined as starting after the CTG trinucleotide (Figure 1). The size and structure of the AFF2 allele are further detailed in the results.

Sequencing of the AFF2 CCG repeat

AFF2 TP-PCR and standard/conventional PCR were performed as described above except that all primers were unlabeled. TP-PCR and standard/conventional PCR products were purified using Nanodrop ^™ 1000 spectrophotometer (Thermo Scientific ^™ , Waltham, Massachusetts, USA). Each 20 μl sequencing reaction contained 10-50 ng of purified standard PCR or TP-PCR product, 1× Terminator Ready Reaction Mix (Applied Biosystems), 2.5x Q-Solution (Qiagen) and 3.2 pmol of AFF2-F primer were added. Initial denaturation was performed at 96°C for 1 minute, followed by 25 cycles of 98°C for 10 seconds, 60°C for 5 seconds and 60°C for 4 minutes. The extension products were purified using Oligo Clean & Concentrator ^TM columns (Zymo Research, Irvine, California, USA) according to the manufacturer's instructions. The eluted purified extension products were concentrated in a Savant The samples were vacuum dried for 5 minutes in a concentrator (Thermo Scientific), resuspended in 12 μl of Hi-Di ^TM formamide (Applied Biosystems), and resolved in a 3130xl genetic analyzer (Applied Biosystems) using a 36 cm capillary filled with POP-7 ^TM polymer. The mixture was electrokinetically injected for 16 seconds at 1.2 kV and electrophoresed at 60° C. for 20 minutes. Post-CE analysis was performed using sequencing analysis software v6.0 (Applied Biosystems).

result

Evaluation of duplex TP-PCR on normal samples and samples positive for FMR1 and AFF2 triplet repeat expansions

The FRAXE folate sensitivity fragile site on the X chromosome is associated with several medical conditions, including intellectual disability, obsessive-compulsive disorder, and premature ovarian failure. FRAXE fragility is caused by an overexpansion of CCG trinucleotide repeats within the 5' untranslated region (UTR) in exon 1 of the AFF2 (formerly FMR2) gene (ClinVar: VCV000010526.1). Overexpansion from the normal allele, which ranges from 6-30 repeats, to >200 repeats is accompanied by repeated CpG methylation. This in turn silences the expression of AFF2, which is a subunit of SEC-L2 that regulates the transcription of several genes. AFF2 CCG repeat overexpansion is the genetic mutation that causes Fragile XE nonsyndromic intellectual disability (FRAXE NSID; OMIM 309548), a mild (IQ 50-70) to borderline (IQ 70-85) intellectual disability estimated to affect one in 50,000 to 100,000 males, as well as other cognitive/behavioral abnormalities including obsessive-compulsive disorder. Paradoxically, alleles with fewer than 11 repeats or with microdeletions within or near the repeats are enriched in patients with premature ovarian failure. Studies linking medium (31-60) repeat sizes to Parkinson's disease, a neurodegenerative motor system disorder, have been inconclusive.

FRAXE shares similar genetic features with the well-studied fragile site FRAXA on chromosome X, which has more clearly demonstrated clinical involvement. The FRAXA site contains CGG repeats within the 5'UTR in exon 1 of the FMR1 gene. Overexpansion of the FMR1 CGG repeats to >200 with accompanying CpG methylation and FMR1 gene silencing results in fragile X syndrome (FXS; OMIM309550) (ClinVar: VCV000009972.1). FXS is the most common inherited monogenic cause of intellectual disability, affecting approximately 1 in 5000 males and 1 in 4000 to 8000 females. Premutation alleles are associated with a number of behavioral traits, including anxiety, obsessive-compulsive disorder, and depression. In addition, approximately one in five females carrying premutation alleles (55-200 CGG repeats) suffer from fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 premutations have been identified in 2% of sporadic POI and 14% of familial POI patients, making FXPOI the most common genetic cause of POI in euploid females. In addition, a subset of FMR1 premutation carriers will eventually develop fragile X-associated tremor/ataxia syndrome (FXTAS), a neurodegenerative disorder characterized by ataxia, tremor, and parkinsonism that affects approximately 1 in 4,000 males and 1 in 7,800 females over the age of 55. The FMR1 gene has also been implicated in Parkinson's disease.

Although both FRAXA and FRAXE fragile sites are closely related to intellectual disability, the mild to critical phenotype of FRAXENSID can cause insufficient determination and insufficient diagnosis compared with FXS. Insufficient diagnosis can be partly attributed to the lack of fast, simple and inexpensive determination to screen the repeat expansion at the FRAXE site. Although the standard PCR method for the repeat expansion at the FRAXE site as an independent determination or multiple determination has been described, they cannot detect large premutation and full mutation alleles, and the detection of these alleles still relies on Southern blot analysis. Because the non-syndromic nature of FRAXE (i.e. lack of characteristic and consistent clinical manifestations, in contrast to the facial deformity (prominent ears, chin, forehead and long face) and macroorchidism present in, for example, FXS patients), molecular diagnosis is crucial for confirming FRAXE.

Initially 12 DNA samples were utilized to evaluate the duplex TP-PCR assay. Figures 2 and 3 show the TP-PCR electropherogram patterns generated from normal males and females and individuals with FMR1 and AFF2 premutation (PM) or full mutation (FM). The leftmost peak generated by the AFF2 TP-PCR reaction is not generated by the annealing of the TP primer within the AFF2 CCG repeat, which begins after the last CTG trinucleotide of the 5' flanking sequence. Instead, this isolated peak that migrates as a distinct 111 bp fragment is generated by the annealing of the TP primer to the (CCG) ₄ CCT (SEQ ID NO:6) sequence just upstream of the repeat (Figure 1). The first true peak generated by the annealing of the TP primer to the CCG 1-5 of the AFF2 repeat appears as a distinct 138 bp fragment on the electropherogram. Annealing of the TP primer occurs at all other positions in the repeat containing five consecutive CCGs (CCGs 2-6, 3-7, etc.), with the final fluorescence peak resulting from annealing of the TP primer to the last five CCGs of the repeat. Thus, the repeat size of the AFF2 allele can be quickly and easily determined by counting the number of peaks arising from within the repeat (i.e., excluding the leftmost isolated peak) and adding four to that number.

The absence of a fluorescence peak that is more than 55 repeats indicates that there is no expansion, while a continuous fluorescence peak that extends more than 55 repeats and up to 200 repeats indicates the presence of a premutation (PM) allele, and a fluorescence peak that extends more than 200 repeats indicates the presence of a full mutation (FM). These results indicate that dual TP-PCR assays successfully detect and accurately identify FMR1 CGG and AFF2 CCG repeat premutations and full mutations in male and female DNA samples (Fig. 2 and Fig. 3).

Population allele distribution and identification of repeat structure

A total of 466 male DNA samples (including DNA from 161 Chinese, 158 Malays, 89 Indians, 44 Caucasians, and 14 African Americans) were genotyped using duplex TP-PCR to determine the allele size distribution (Figure 4 and Table 2). Different allele distributions were observed among ethnic groups, with the modal AFF2 repeat size being 18 for Chinese and Malays (32.3% and 27.8%, respectively), but 15 for Caucasians (40.9%), African Americans (50.0%), and Indians (32.6%). The allele size range was wider in Chinese (5-31) and Malays (6-37) compared to Indians (10-27), Caucasians (9-26), and African Americans (14-28), which may be partly attributed to the larger sample size of Chinese and Malays. Using conventional classification, 24 samples had the smallest allele (<11 repeats), 440 samples had the normal allele (11-30 repeats), and the remaining 2 samples had the intermediate allele (31-60 repeats). No premutation and full mutation alleles were observed. These results are similar to early studies on the distribution of AFF2 alleles in Chinese Han (mode 18, range 9-26) and Caucasians in New York City (mode 16, range 8-34) (Figure 4A-C).

The chromosome carrying 29 FMR1 CGG repeats and 18 AFF2 CCG repeat combinations is the most common (Fig. 4D, upper figure). In the 466 male population DNA samples screened, three AFF2 TP-PCR electrophoresis patterns were observed. As recorded in GenomeReference Consortium Human Build 38 (GRCh38), the most common pattern was present in 459 samples (98.1%), which represented most normal and all neutral alleles, and the other two patterns were identified by Sanger sequencing as being caused by the presence of two single nucleotide polymorphism (SNP) variants (Fig. 4D, lower figure).

One of the SNP variants, a T>C substitution at chrX:148,500,637 (rs868914124, GRCh38), converts the CTG trinucleotide to CCG at the 5' start of the AFF2CCG repeat. Although the AFF2 trinucleotide repeat in the rs868914124 (T) common allele starts after the last CTG trinucleotide of the 5' flanking sequence (Figure 5A), this CTG trinucleotide becomes a CCG trinucleotide in the rs86891412 (C) variant allele, thereby extending the AFF2 trinucleotide repeat segment upstream by 2 CCGs or 6 bp to start after the last CAG trinucleotide of the 5' flanking sequence (Figure 5B). Therefore, the first TP-PCR product produced in the repeat segment of rs868914124 (C) variant allele appears as an obvious 132bp fluorescence peak (Fig. 5 B), contrasted with the 138bp first fluorescence peak (Fig. 5 A) from rs868934124 (T) common allele. In the TP-PCR electrophoretogram, this size difference is shown as a narrower gap (Fig. 5 B) of 20bp between the leftmost isolated peak and the first peak of the AFF2 CCG of rs868914124 (C) variant allele compared with the 26bp wider gap (Fig. 5 A, C) of rs868914124 (T) common allele.

rs868914124 (C) variant was observed in 8 (1.72%) of 466 AFF2 alleles (Fig. 4D, lower figure, row B), of which 6 were Malay alleles. Compared with 21 of 458 samples carrying rs868941424 (T), 3 of 8 samples with rs868914124 (C) variant contained minimal alleles (<11 repeats). Using Fisher's exact test (two-tailed), it was observed that this rare variant was enriched in the Malay group (odds ratio = 6.01; 95% confidence interval 1.06-61.7; P = 0.021), and it had minimal alleles (odds ratio = 12.3; confidence interval 1.79-68.4; P = 0.006). Interestingly, all six AFF2 expanded alleles (five full mutations and one premutation) carried the rare rs868914124(C) variant, although the expanded alleles from the affected male and his uncle, as well as from the father-daughter pair, were considered identical in ancestry (Fig. 5D and Figs. 6 and 7).

Unlike the AGG interruptions within the CGG repeats commonly observed in normal FMR1 alleles, we observed only one normal AFF2 allele (0.21%) containing a non-CCG interruption within its repeat segment, namely a CTG interruption at the fifth repeat position (Figure 4D, bottom panel, row C and Figure 5C). This interruption was caused by a C>T substitution at chrX:148,500,652 (rs1389911365, GRCh38). Annealing of the TP primer at a position that includes this interruption will result in mismatched pairing, and no PCR product can be generated from this position. In the TP-PCR electropherogram, failed PCR at the primer mismatch position appears as a gap without a peak (Figure 5C).

Interestingly, new non-CCG interruptions were also observed in all 6 AFF2 expansion positive samples. Based on the TP-PCR electropherogram patterns of the four AFF2 FM males (which show gaps with or without peaks/missing peaks between peak clusters), it was initially suspected that the interruptions were within the repeat (Figure 3). Sanger sequencing revealed that there were one or more non-CCG interruptions at the 5' end of the repeat, which carried the same sequence CCTGTGCAG as a 9-nucleotide section 5' just upstream of the repeat. The number of interruptions varied from one (Figure 6A) to more than four (Figure 6B). Their positions also varied, for example from the 8th to the 10th repeat position (Figure 6A) or from the 6th to the 8th position (Figure 7).

We observed that in the father-daughter pair (Figure 7), the AFF2 CCG repeat expansion was a full mutation in the father (DNA_25926) and a premutation in the daughter (DNA_3802), indicating a previously documented contraction at transmission. Although the AFF2 full mutation alleles of the nephew (FX0229) and uncle (FX0230) (Figure 6) were considered identical in ancestry, their repeat structures differed due to the presence of an additional non-CCG interruption in the uncle. The exact cause or mechanism of this difference has not been investigated.

Blind validation of the duplex TP-PCR assay on previously characterized samples

82 archived and previously characterized genomic DNAs were included in the blind validation of the duplex TP-PCR assay. The AFF2CCG repeat was sized as described in Materials and Methods. The duplex TP-PCR assay accurately classified all 40 normal, 17 FMR1 PM, 23 FMR1 FM, and 2 AFF2 FM samples included in the test (Table 3).

Table 2. AFF2 CCG repeat allele frequencies in five populations.

CH, Chinese; ML, Malay; IN, Indian; CAU, Caucasian; AA, African American

Table 3. Concordance analysis of samples with or without FMR1 or AFF2 repeat expansions.

Example 2: Spinocerebellar Ataxia (SCA)

Materials and methods

Biological samples

Genomic DNA and cell lines were obtained from Coriell Cell Repositories (CCR; Coriell Institute for Medical Research, Camden, NJ, USA). NA06926, NA13536 and NA13537 each carry an expanded ATXN1 CAG repeat, NA14982 carries an expanded ATXN2 CAG repeat, GM06151 carries an expanded ATXN3 CAG repeat, NA03562 carries an expanded ATXN7 CAG repeat, and NA13716 and NA13717 each carry an expanded ATN1 CAG repeat. GM16243 carries an expanded FXN GAA repeat, GM06907 carries an expanded FMR1 CGG repeat, and GM05164 and GM06075 each carry an expanded DMPK CTG repeat. Two clinical DNA samples 160920 and 183254 carrying expanded CACNA1A CAG repeats and PPP2R2B CAG repeats, respectively, were obtained from KK Women’s and Children’s Hospital. Sixty archival DNA samples with known genotypes were obtained from Siriraj Hospital-Mahidol University and included in the blinded evaluation of assay accuracy.

Seven-plex TP-PCR

Seven TP-PCRs were performed in a 25 μl reaction containing: 10 ng of genomic DNA, 1.5× Q-Solution (Qiagen, Hilden, Germany), ₁ × PCR buffer (Qiagen) containing 1.5 mmol/L of MgCl, a mixture of deoxyribonucleic acid triphosphates (dNTPs) (Roche Applied Science, Penzberg, Germany) and 2 units of HotStar Taq DNA polymerase (Qiagen) consisting of dATP, dTTP, dCTP and dGTP of each 0.2 mmol/L. Eight primers (seven fluorescently labeled locus-specific primers and one universal TP primer TP-R) were included at their respective optimal working concentrations. Primer sequences, fluorophore labels and primer concentrations are shown in Tables 4 and 5. Thermal cycling consisted of initial polymerase activation at 95°C for 15 min, followed by 35 cycles of 98°C for 45 s, 60°C for 1 min, and 72°C for 2 min, and a final extension at 72°C for 5 min on a SimpliAmp thermal cycler (Applied Biosystems-Thermo Fisher Scientific, Foster City, CA, USA).

Capillary electrophoresis (CE)

The aliquot of 1-μ L fluorescently labeled TP-PCR product is mixed with the Hi-Di ^TM formamide (Applied Biosystems) of 9 μ L and the GeneScan ^TM 500LIZ ^TM dye size standard (Applied Biosystems) of 0.5 μ L, then it is denatured at 95 ℃ for 5 minutes, quickly cooled to 4 ℃, and resolved in the 3130xl genetic analyzer (Applied Biosystems) of the 36-cm capillary that uses to be filled with POP-7 ^TM polymer.Sample is injected electrically for 15 seconds under 1kV, and electrophoresis is carried out for 40 minutes at 60 ℃.GeneScan is analyzed using GeneMapper 5.0 software (Applied Biosystems).The amplified product from each locus can be identified by its product size range and peak color.Sevenfold TP-PCR product shows the electrophoresis peak of four different colors, and can be analyzed together by opening all four fluorescence detection channels, or use a fluorescence detection channel to close other channels simultaneously and analyze respectively.

result

Detection of expanded CAG repeats by single-tube seven-plex TP-PCR

Spinocerebellar ataxia (SCA) is a neurodegenerative disease that causes degeneration of the cerebellum and sometimes the spinal cord and is primarily characterized by ataxic gait, poor hand-eye coordination, and slurred speech. These autosomal dominant disorders are genetically heterogeneous and can be caused by conventional mutations as well as repeat expansion mutations. Overall, the prevalence of SCAs worldwide averages 2.7 cases per 100,000 individuals, with SCA3 being the most common. There are more than 40 genetically distinct SCAs, at least 12 of which are caused by repeat expansions. Of these, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, and SCA17 are caused by abnormal CAG trinucleotide repeat expansions. Another disease, dentatorubral pallidum-Lewy body atrophy (DRPLA), is also caused by CAG repeat expansions and is classified as an SCA due to its heterogeneity and overlapping clinical phenotypes with other SCAs.

SCA1 is caused by a CAG repeat expansion in exon 8 of the ATXN1 gene on chromosome 6p22.3. Non-expanded (normal) ATXN1 alleles contain 6 to 44 CAGs interrupted by CAT. When not interrupted, alleles containing 36 to 38 CAGs are variable normal alleles that may expand into the pathogenic size range when transmitted to the next generation, but are not associated with clinical symptoms per se, while those containing ≥39 CAGs are pathogenic. SCA2 is caused by a CAG repeat expansion in exon 1 of the ATXN2 gene on chromosome 12q24.12. Normal ATXN2 alleles contain 14 to 31 pure or CAA interrupted CAGs, and those containing 32 CAGs are intermediate alleles of uncertain clinical significance. Alleles containing 33 to 500 CAGs are pathogenic. SCA3, or Machado-Joseph disease, is caused by a CAG repeat expansion in exon 10 of the ATXN3 gene on chromosome 14q32.12. Normal ATXN3 alleles contain 12 to 44 CAGs, while those containing 45 to 59 CAGs are intermediate alleles that are susceptible to expansion, and those with 60 to 87 CAGs are full-penetrance alleles. SCA6 is caused by a CAG repeat expansion in exon 47 of the CACNA1A gene on chromosome 19p13.13. Normal CACNA1A alleles contain ≤18 CAGs, while full-penetrance alleles contain 20 to 33 CAGs. CACNA1A alleles containing 19 CAGs are of uncertain clinical significance. SCA7 is caused by a CAG repeat expansion in exon 3 of the ATXN7 gene on chromosome 3p14.1. Normal ATXN7 alleles contain 7 to 27 CAGs, while those containing 28 to 33 CAGs are variable normal alleles, and allelic mutations with ≥34 CAGs are pathogenic. SCA12 is caused by a CAG repeat expansion in the 5' region of the PPP2R2B gene on chromosome 5q32. Normal PPP2R2B alleles contain 7 to 32 CAGs, while those containing 51 to 78 CAGs are full-penetrance alleles. DRPLA is caused by a CAG repeat expansion in exon 5 of the ATN1 gene on chromosome 12p13.31. Normal ATN1 alleles contain 6 to 35 CAGs, while those containing ≥48 CAGs are full-penetrance alleles.

The various SCA types often present with distinct phenotypes and are often difficult to distinguish by signs and symptoms alone due to extensive clinical overlap and other concomitant non-ataxia phenotypes. Therefore, molecular genetic testing is necessary and is the only way to identify the causative mutation to confirm disease status in symptomatic individuals. Identification of causative genes can be time-consuming and expensive, as some genes are difficult to test in the absence of a known family history or disease-specific symptoms. The European Molecular Genetics Quality Network recommends that all laboratories should offer testing for the five most common SCA types (SCA1, SCA2, SCA3, SCA6, and SCA7) as a minimum, while testing for other types will depend on local prevalence.

Detection and sizing of CAG repeats in different SCAs relies on standard PCR or three-primer PCR (TP-PCR) followed by capillary electrophoresis. Repeat sizing by standard PCR must typically be accompanied by low-throughput and labor-intensive Southern blot analysis to confirm the presence of very large alleles and estimate their repeat size, or by enzymatic digestion to determine the presence of CAT interruptions in expanded ATXN1 alleles, when only a single fragment from the normal allele is detected. TP-PCR, first described by Warner et al. [24], has been widely used to detect repeat expansions that cause many repeat expansion diseases, regardless of the size of the expansion. It uses locus-specific flanking primers, a three-primer (TP) primer, and a tail primer and is able to detect very large expansions, accurately size all normal alleles and modest expansions, and identify interruptions within the repeat segment through distinct TP-PCR electropherogram patterns.

Because multiple rounds of genetic testing may be required before the causative gene is identified in SCA patients, simultaneous screening of trinucleotide repeats in different disease genes may facilitate faster identification of the causative gene in addition to saving the cost of multiple testing. We report the development of a new single-tube multiplex TP-PCR assay that enables simultaneous screening of expanded mutations at seven loci responsible for some of the most common SCAs (SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, and DRPLA). The seven-plex assay uses locus-specific flanking primers that differentially label each repeat locus, as well as a universal primer that anneals within CAG.

The SCA seven-plex TP-PCR assay utilizes fluorescently labeled locus-specific flanking primers located upstream of the CAG repeats of each disease gene and a universal TP primer that anneals within all CAG repeats to enable CAG repeats from seven different SCA loci to be co-amplified in a single reaction tube. Each flanking primer is labeled with one of four fluorophores (Ned, Vic, Fam, or Pet). In addition, each flanking primer is at a different distance from its respective repeat segment so that the electrophoretic peak generated from a normal allele of one repeat locus does not overlap with the electrophoretic peak of a normal allele from another repeat locus labeled with the same fluorophore (Figure 8). The combined effect of differential fluorophore labeling and positioning of the seven flanking primers is that the TP-PCR products generated by the normal alleles of each repeat locus do not overlap and can be distinguished after capillary electrophoresis. The number of repeats is determined by counting the electrophoretic peaks starting from the left side of the electropherogram, with the first peak representing the first five pure CAGs of the repeat segment (Figure 8).

The seven-plex TP-PCR assay was first tested on samples of known genotype to confirm locus-specific annealing of each flanking primer and detection of extended CAG repeats at each extended repeat locus. When the TP primer anneals to five consecutive CAG repeats, TP-PCR produces a mixture of fragments, each of which differs by one triplet. The minimum amplification product of all loci contains five CAG triplets, except ATXN3, which has 11 triplets (Figure 8A) due to the presence of CAA at the 3rd and 6th triplet positions and AAG at the 4th triplet position in the wild-type allele of ATXN3. The electropherogram shows a series of continuous ladder peaks (Figure 8B) because the TP primer anneals to multiple positions within the uninterrupted CAG repeat (Figure 8A). Each continuous electrophoretic peak represents a TP-PCR product that is one repeat or three base pairs larger, and the repeat size in the allele can be derived from the number of counted peaks. The presence of non-CAG interruptions in the middle of the repeat segment that may occur in the ATXN1 and ATXN2 alleles prevents the TP-polymers from effectively annealing at these positions, resulting in the absence of fluorescence peaks between the peak clusters. The extended negative sample produces a TP-PCR product produced by two non-extended (normal) alleles within the normal repeat size range (Figure 9), while the extended positive sample additionally produces a longer TP-PCR product produced by the extended allele (Figure 10). Samples whose repeat size exceeds the upper limit of the normal allele size range at a specific repeat locus will indicate an expansion at the repeat locus (Figure 10).

Blind clinical sample validation

In order to assess the ability of seven TP-PCR assays to accurately identify the expansion of seven repetitive loci in affected patient samples, 60 archived clinical DNA samples with known genotypes were blindly analyzed. After TP-PCR and capillary electrophoresis, a fluorescence detection channel was opened once, so that the amplified products labeled with different fluorophores were analyzed respectively. This enables the sample of the electrophoresis peak that demonstrates the upper limit of the normal allele size range at any one of the seven SCA loci to be clearly identified. For all 31 DNA samples (5 SCA1 positives, 7 SCA2 positives, 12 SCA3 positives, 5 SCA6 positives, 1 SCA7 positives and 1 DRPLA positive) that are positive for one of the seven SCA repetitive expansions, seven TP-PCRs correctly detected the repetitive expansion (table 6) in the expected SCA repetitive loci. Single PCR involving the flanking primers and universal TP primers of the relevant repetitive loci was carried out on all screening positive samples to confirm the result (data not shown). No expansion was detected in any one of the 29 negative DNA samples of expansion.

Table 4. SCA seven-plex TP-PCR primers and expected TP-PCR product size

Table 5. Expected TP-PCR product sizes for SCA sevenfold

Table 6: Disease status and CAG repeat size for 60 archived DNA samples with known genotypes

References

Zhong, N. et al., A survey of FRAXE allele sizes in three populations. Am JMed Genet, 1996.64(2):p.415-9.

Claims (21)

1. A method for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject, the method comprising: i) contacting the nucleic acid sample with: a) gene-specific primers that specifically bind to different target sequences of each gene, wherein the gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence, and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primers; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; and ii) analyzing the amplification product. 2. The method of claim 1, wherein the method comprises analyzing the amplification product using a size separation technique or a sequencing technique. 3. The method of claim 2, wherein the size separation technique is an electrophoresis-based technique. 4. The method according to any one of claims 1 to 3, wherein the amplification products are separated according to size. 5. The method according to any one of claims 1 to 4, wherein a size change of a gene amplification product compared to a reference indicates the presence of a repeat expansion sequence in the gene. 6. The method according to any one of claims 1 to 5, wherein the repeat expansion sequence is a trinucleotide repeat expansion sequence. 7. The method according to any one of claims 1 to 6, wherein the trinucleotide repeat sequence is selected from (CGG) _n , (CCG) _n , (CAG) _n , (CTG) _n , (GCC) _n , (GGC) _n , (GAA) _n or (TTC) _n , wherein n is 2 to 200 or more. 8. The method according to any one of claims 1 to 7, wherein the universal primer comprises a unique 5' tail sequence. 9. The method of claim 8, wherein the method comprises providing a tail primer that specifically binds to a unique 5' tail sequence of the universal primer. 10. The method according to any one of claims 1 to 9, wherein the universal primer binds to a common target sequence comprising or consisting of 5, 6, 7, 8, 9, 10 or more consecutive trinucleotide repeats. 11. The method according to any one of claims 1 to 10, wherein the gene-specific primer is labeled. 12. The method according to any one of claims 1 to 11, wherein the two or more genes consist of or comprise FMR1 and AFF2. 13. The method of claim 12, wherein the method comprises contacting the nucleic acid sample with: a) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 1; b) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 2; and c) a universal primer comprising or consisting of a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of (CGG) ₅ (SEQ ID NO: 15) or SEQ ID NO: 3. 14. The method of any one of claims 1 to 11, wherein the two or more genes are selected from SCA1, SCA2, SCA3, SCA6, SCA7, SCA12 and DRPLA. 15. The method of claim 14, wherein the method comprises contacting the nucleic acid sample with: a) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 7; b) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 8; c) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 9; d) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 10; e) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 11; f) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 12; and/or g) a gene-specific primer comprising or consisting of a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, and h) a universal primer comprising or consisting of a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of (CTG) ₅ (SEQ ID NO: 16) or SEQ ID NO: 14. 16. A kit for detecting the presence or absence of a repeat expansion sequence in two or more genes in a nucleic acid sample obtained from a subject, the kit comprising: a) a gene-specific primer that specifically binds to a different target sequence in each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequence is upstream or downstream of the nucleotide repeat sequence in each gene; and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer for each gene; The gene-specific primers and the universal primers are capable of generating one or more amplification products from each gene. 17. A composition comprising a nucleic acid sample obtained from a subject, the composition comprising a) gene-specific primers that specifically bind to different target sequences of each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primer of each gene; The gene-specific primers and the universal primers are capable of generating one or more amplification products from each gene. 18. A method of screening a subject for one or more multiple repeat expansion diseases, the method comprising: i) contacting a nucleic acid sample from the subject with: a) gene-specific primers that specifically bind to different target sequences of each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequences are upstream or downstream of the nucleotide repeat sequence in each gene; and b) universal primers that bind to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primers and the universal primer are capable of producing one or more amplification products from each gene; and ii) analyzing said amplification products to screen said one or more multiple repeat expansion diseases in said subject. 19. The method of claim 18, wherein the one or more multiple repeat expansion diseases comprise or consist of fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI), fragile X-associated tremor/ataxia syndrome (FXTAS), and fragile XE non-syndromic intellectual disability (FRAXE NSID). 20. The method of claim 18, wherein the one or more multiple repeat expansion diseases are spinocerebellar ataxia (SCA) and/or dentatorubral pallidum atrophy with Lewy bodies (DRPLA). 21. A method of screening for one or more multiple repeat expansion diseases in a subject and treating the subject, the method comprising: i) contacting a nucleic acid sample from the subject with: a) a gene-specific primer that specifically binds to a different target sequence for each of two or more genes, wherein each gene comprises a nucleotide repeat sequence, and wherein the different target sequence is upstream or downstream of the nucleotide repeat sequence in each gene; and b) a universal primer that binds to a common target sequence shared by the two or more genes, wherein the common target sequence is located within the nucleotide repeat sequence and is located on the opposite strand to that bound by the gene-specific primer of each gene; wherein the gene-specific primer and the universal primer are capable of producing one or more amplification products from each gene ii) analyzing the amplification products to screen for the one or more multiple repeat expansion diseases in the subject; and iii) treating a subject found to have at least one multiple repeat expansion disorder.