Abstract:
A method of screening a subject for mitochondrial dysfunction comprises detecting the presence or absence of single nucleotide changes in a hypervariable region of the mitochondrial DNA of said subject, the presence of such changes indicating that said subject is afflicted with or at risk of developing mitochondrial dysfunction.

Description:
RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application Serial No. 60/184,379, filed Feb. 23, 2000, the disclosure of which is incorporated by reference herein in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to methods of screening subjects for mitochondrial dysfunction. 
     BACKGROUND OF THE INVENTION 
     A wide variety of clinical manifestations are due to mutations in mitochondrial DNA, but are difficult to diagnose due to the varied clinical picture and the lack of sensitive or specific diagnostic testing. Past efforts to document mtDNA mutations in children believed to have mitochondrial disorders have been hampered by the size of the mitochondrial genome and the presence of numerous benign polymorphisms. 
     Mitochondria are eukaryotic cytoplasmic organelles where oxidative phosphorylation takes place, and are often termed the ‘power plant’ of the cell. In animal cells, the mitochondria is the only cytoplasmic organelle that contains DNA. Human mitochondrial DNA (mtDNA) is a circular molecule of about 16,600 nucleotide pairs which encode thirteen of the at least 82 protein subunits of the complexes in the oxidative phosphorylation pathway, both ribosomal RNAs, and all of the 22 transfer RNAs required for mitochondrial protein synthesis. However, the majority of proteins located in the mitochondria are encoded by nuclear DNA (chromosomal DNA) and translated by cytoplasmic ribosomes, and then imported to the mitochondria. Therefore, a “mitochondrial disorder” can be secondary to a mutation in either the nuclear DNA or in the mitochondrial DNA. 
     The entire human mitochondrial DNA (mtDNA) sequence has been determined (see MITOMAP: Human Mitochondrial Genome Database, Center for Molecular Medicine, Emory University, Atlanta, Ga., USA (1998); Wallace et al. (1995) Report of the committee on human mitochondrial DNA, In: Cuticchia A J (Ed)  Human gene mapping  1995: A Compendium, Johns Hopkins University Press, Baltimore, pp 910-954 (1995)). 
     Mitochondrial genetics differ from nuclear (standard or Mendelian) genetics. Virtually all the mtDNA of a zygote is derived from the oocyte, and mtDNA disorders are transmitted by maternal inheritance. Maternal-linked (matrilineal) relatives presumably have identical mtDNA sequences, except perhaps at the site of a new mutation. Additionally, the mtDNA mutation rate is substantially higher than that of the nuclear DNA. Most cells contain hundreds to thousands of mitochondria, and each mitochondria contains several copies of mtDNA, resulting in high mtDNA copy number. In normal individuals, essentially all of the mtDNA molecules are identical (homoplasmy). However, if there is a mutation in mtDNA, the mutant mtDNA and the normal (wild type) mtDNA often coexist in the same cell or tissue (heteroplasmy). 
     Because of the high mutation rate, mtDNA has numerous polymorphisms. Almost always these polymorphisms are homoplasmic. In contrast, most recognized pathogenic mtDNA mutations are heteroplasmic, especially when disease manifests during childhood (Shoffner and Wallace (1995) In:  The Metabolic and Molecular Basis of Inherited Disease  (7 th  Ed.), New York, McGraw Hill, 1535-1629). 
     As a result of segregation in the pre-oocyte stage, each ova of an affected woman has a different proportion of mutant versus normal mtDNA, which can range from virtually 0 to 100%. Each of her children, therefore, will inherit differing amounts of mutant mtDNA. In addition, normal and mutant mtDNA randomly segregate during the cell divisions of embryogenesis, resulting in different proportions of mutant mtDNA residing in different tissues. The presence of clinical disease in a given tissue is dependent on the specific mutation, the percent of mutant mtDNA and the threshold for that tissue. The percentage of mutant mtDNA necessary to cause clinical symptoms varies from tissue to tissue; for example, 80% mutant mtDNA may be clinically silent in liver but cause symptoms in tissues with higher energy requirements, such as muscle or brain (Shoffner et al. (1991)  Adv. Hum. Genet.  19:267). Since the mutant mtDNA load varies between matrilineal family members, as well as between tissues within each individual, the clinical manifestations of a mtDNA mutation vary widely among affected family members. Healthy family members with mutant mtDNA levels below threshold are common. These individuals, if female, are ‘carriers’ as their children will inherit their mitochondria and, if inherited mutant mtDNA levels are above threshold, the children will be affected. A well known example is the A3243G mtDNA mutation, in which family subjects exhibit variable manifestations, ranging from stroke (usually associated with relatively higher degrees of mutant heteroplasmy) to those (with lesser mutant loads) with diabetes, deafness, or asymptomatic carriers. This phenomenon of varied clinical presentation has been observed with other mtDNA mutations as well. 
     As the mtDNA mutation rate is high, mtDNA disorders may be due to new mtDNA mutations; in such cases matrilineal relatives will be unaffected. In other cases, mothers harbor small degrees of mutant heteroplasmy and are clinically normal or only mildly affected. In a minority of cases, multiple matrilineal relatives harbor various amounts of mutant mtDNA in their tissues and exhibit varying clinical manifestations. 
     A broad spectrum of disease manifestations has been associated with systemic mtDNA mutations. These mutations can be either single point mutations, or large rearrangements (deletions and/or duplications). Rearrangements usually are spontaneous, although they may be maternally inherited or mendelianly inherited secondary to predisposing nuclear mutations. 
     Clinical mitochondrial dysfunction may be defined as idiopathic neuromuscular and/or multisystem disease, biochemical signs of energy depletion, and lack of another diagnosis. Mitochondrial disorders are evidenced when the cellular supply of energy is unable to keep up with demand; symptoms predominate in tissues with the highest energy requirements (brain and muscle). Mitochondrial disorders are most commonly displayed as neuromuscular disorders, including developmental delay, seizure disorders, hypotonia, skeletal muscle weakness and cardiomyopathy. Other manifestations which have been reported include gastroesophageal reflux, apnea, optic atrophy, deafness, acute liver failure, diabetes mellitus, and other hormonal deficiencies. 
     Mitochondrial disorders are often not suspected until late in a diagnostic work-up. Confirmation of a mitochondrial disorder is, at present, a time-consuming and expensive process, and may include lactic acid measurement in body fluids and diagnostic muscle biopsy for electron microscopy and assay of the electron transport chain activities in vitro. However, these methods rarely specify the mode of inheritance or allow for presymptomatic or prenatal diagnosis. 
     SUMMARY OF THE INVENTION 
     A method of screening a subject for mitochondrial dysfunction is disclosed. The method comprises detecting the presence or absence of single nucleotide changes in a hypervariable region of the mitochondrial DNA of said subject, the presence of such changes indicating that said subject is afflicted with or at risk of developing mitochondrial dysfunction. 
     Also disclosed is the use of a means for detecting the presence or absence of single nucleotide changes in a hypervariable region of the mitochondrial DNA of a subject in or for determining if that subject is afflicted with or at risk of developing mitochondrial dysfunction. 
     The foregoing and other objects and aspects of the present invention are explained in greater detail below. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows the pedigrees in patients 1 and 8. Each of the disease manifestations present in these families is consistent with a possible mitochondrial disorder. Squares=males; circles=females; and circles within squares=either gender. Arabic numbers within symbols indicate the number of (normal) individuals. The arrows point to our cases (probands). 
     FIG. 2 shows an expanded diagram of human mtDNA-CR. The mtDNA-CR is defined as the area flanked by the tRNA genes for proline (tRNA Pro ) and phenylalanine (tRNA Phe ) (boxes with diagonal stripes). The location of each heteroplasmic nucleotide found among the cases described herein are marked by black lines and by the nucleotide number. Although not precisely defined, the approximate locations are noted for the hypervariable regions (HV1 and HV2, box with vertical stripes), evolutionary conserved sequence blocks (CSBs, solid boxes), displacement loop (D-loop, open box), the origin of heavy strand replication (O H ), and the transcription initiation sites for the 2 heavy strand (H1 and H2) and light strand (L) transcripts (arrows indicating the direction of transcription). The cross-hatched boxes at the bottom of the figure depict the areas screened by each of the PCR-amplified segments described herein. 
     FIG. 3 shows a computer scanned image of a TTGE gel of the 7S segment in 5 unrelated individuals: a normal control (lane 1), a child with length heteroplasmy at 16184 (lane 2), two subjects with different point heteroplasmic variants (lanes 3 &amp; 4), and rho negative cell culture (lane 5). As shown here, in the absence of heteroplasmy (homoplasmy) a single distinct band is seen on the gel, while heteroplasmic samples usually demonstrate 3 or 4 bands on TTGE, corresponding to the predicted 2 homoduplex and 2 heteroduplex species (often the homoduplex bands are not separated). 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     The present invention is primarily intended to be carried out on human subjects, including both male and female subjects, of any age, including juvenile subjects. The method may be carried out on subjects who have been previously diagnosed as afflicted with mitochondrial dysfunction, or as a prognostic test on subjects who have not yet been diagnosed as afflicted with mitochondrial dysfunction. The test may be carried out as a screening procedure, wherein a positive result in the test indicates increased risk of mitochondrial dysfunction. 
     The mtDNA hypervariable region from which changes or mutations indicative of mitochondrial dysfunction generally consists of hypervariable region HV1 and HV2. Any change or mutation set forth in EXAMPLES hereto may be used in carrying out the test. 
     The changes or mutations described herein may be detected by any suitable technique, including but not limited to DNA amplification techniques such as described in U.S. Pat. No. 5,767,248 to Roses et al., the disclosure of which is incorporated herein by reference, adapted to be carried out on the changes or mutations described herein. 
     EXAMPLE 1 
     Inheritance of Mitochondrial Disorders 
     Children with idiopathic neuromuscular and/or multi-system disease manifestations are frequently encountered in pediatric tertiary practice. Some of these children bear one or more descriptive diagnoses such as mental retardation, epilepsy, migraine or cardiomyopathy. Increasingly, a proportion of these children are found to have biochemical signs of a possible defect in energy metabolism such as an elevated body fluid lactate, abnormal urine organic acids and/or mitochondrial proliferation by enzyme analysis in muscle. Whether these findings represent primary inborn errors of mitochondrial energy metabolism (‘mitochondrial disorders’) or non-specific secondary phenomenon remains controversial. 
     It has been observed that many of these children have matrilineal relatives with widely different, often transient and mild, neuromuscular and/or multi-system manifestations, including migraine (FIG.  1 ). This observation suggests that a maternally-transmitted factor may be involved in the development of disease manifestations in a substantial proportion of cases. Since mitochondria contain their own DNA (mtDNA) which is maternally transmitted (Shoffner and Wallace (1995) Oxidative phosphorylation diseases. In: Scriver C R, Beaudet A L, Sly W S, Valle D, editors.  The metabolic and molecular bases of inherited disease , 7th ed. New York: McGraw-Hill, pg 1535-629; Chinnery and Turnbull (1997)  Q J Med,  190:657-66), mtDNA sequence variations likely constitute this maternally-transmitted factor. However, known mtDNA mutations are infrequently (&lt;5%) identified in children suspected of having a mitochondrial disorder (Liang and Wong (1998)  Am J Med Genet , 77:395-400), suggesting that additional pathological mtDNA variants remain to be discovered. 
     Mitochondrial genetics differs in many aspects from Mendelian, or nuclear, genetics. Mitochondrial DNA is a 16,569 base pair circular molecule which encodes 13 protein subunits of the mitochondrial respiratory chain, as well as the 22 transfer RNA and 2 ribosomal RNA genes required for the translation of the mtDNA-encoded proteins (Shoffner and Wallace (1995) Oxidative phosphorylation diseases. In: Scriver C R, Beaudet A L, Sly W S, Valle D, editors.  The metabolic and molecular bases of inherited disease,  7th ed. New York: McGraw-Hill, pg 1535-629). Mitachondrial DNA exists at high copy number, usually on the order of hundreds or thousands of genomes per cell. Heteroplasmy, defined as the existence of two or more different mtDNA sequences in the same cell, is associated with several, predominantly neuromuscular and/or multi-system disease states in man (Shoffner and Wallace (1995) Oxidative phosphorylation diseases. In: Scriver C R, Beaudet A L, Sly W S, Valle D, editors.  The metabolic and molecular bases of inherited disease,  7th ed. New York: McGraw-Hill, pg 1535-629; Chinnery and Turnbull (1997)  Q J Med,  190:657-66). Over 80 different pathological mtDNA point mutations have been described to date, and the vast majority exist in patients in a heteroplasmic state along with wild-type genomes (Shoffner and Wallace (1995) Oxidative phosphorylation diseases. In: Scriver C R, Beaudet A L, Sly W S, Valle D, editors.  The metabolic and molecular bases of inherited disease,  7th ed. New York: McGraw-Hill, pg 1535-629; Kogelnik et al. (1996)  Nuc Aci Res,  24:177-9. In contrast, normally occurring mtDNA point sequence variants (polymorphisms) are almost always homoplasmic (only one mtDNA sequence present) (supra). Clinical symptoms occur once the proportion of mutant mtDNA molecules exceeds a threshold which is both tissue and mutation specific (Shoffner and Wallace (1995) Oxidative phosphorylation diseases. In: Scriver C R, Beaudet A L, Sly W S, Valle D, editors.  The metabolic and molecular bases of inherited disease,  7th ed. New York: McGraw-Hill, pg 1535-629; Chinnery and Turnbull (1997)  Q J Med,  190:657-66). Partially in response to varying proportions of mutant and wild-type heteroplasmy among different tissues within each individual and among different family members, maternal inheritance is often characterized by widely variable clinical manifestations, age of onset and severity among members of the same family (supra). 
     As the only large non-coding region in the mitochondrial genome, the approximate 1 kilobase (kb) mtDNA control region (CR) is involved in mtDNA replication, transcription and membrane attachment (FIG. 2) (Shoffner and Wallace (1995) Oxidative phosphorylation diseases. In: Scriver C R, Beaudet A L, Sly W S, Valle D, editors.  The metabolic and molecular bases of inherited disease,  7th ed. New York: McGraw-Hill, pg 1535-629; Kogelnik et al. (1996)  Nuc Aci Res,  24:177-9. Control region mutations which interfere with these and other ‘regulatory’ functions constitute a plausible mechanism for human disease, although disease-causing mutations in this region have not been reported. Within the mtDNA-CR are 2 highly polymorphic regions, termed hypervariable region 1 and 2 (HV1, HV2) (supra). A very common HV1 variant consisting of an expanded homopolymeric tract of cytosines (length heteroplasmy) was recently reported as associated with insulin resistance (Poulton et al. (1998)  Diabetologia,  41:54-8). The present study shows the presence of multiple heteroplasmic and homoplasmic single nucleotide changes in the hypervariable regions of the mtDNA-CR in 13 of 67 children with idiopathic neuromuscular and/or multi-system disease manifestations and an elevated body fluid lactate concentration. 
     EXAMPLE 2 
     Case Reports 
     Clinical and laboratory findings in each of the 11 children with mtDNA-CR point heteroplasmy patterns on temporal temperature gradient gel electrophoresis (TTGE) are listed in Table 1. Although there is substantial variability in clinical manifestations among the 11 children as a whole, many cases cluster into clinically-based groups, some of which may be syndromic. The following 6 cases were chosen for a more detailed description in order to demonstrate the range of phenotypic expression. Patient 8 was previously reported in brief (Boles and Williams (1999)- Dig Dis Sci,  44 (Suppl.):103S-107S. Pedigrees for patients 1 and 8 are shown in FIG.  1 . 
     
       
         
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
             
             
               
                   
                 Patient #1 
                 Patient #2 
                 Patient #3 
               
               
                   
               
               
                 Brief Clinical 
                 Reversible severe 
                 Reversible severe 
                 Reversible severe 
               
               
                 Description 
                 cardiomyopathy 
                 cardiomyopathy 
                 cardiomyopathy 
               
               
                 Age/Sex/Race 
                 7/M/B 
                 4/F/H 
                 2/F/H 
               
               
                 Heteroplasmy 
                 16168C-T 
                 Insufficient DNA 
                 16093T-C 
               
               
                 Growth 
                 Failure-to-thrive, 
                 Early 
                 Short stature 
               
               
                   
                 Short stature 
                 failure-to-thrive 
               
               
                 Developmental 
                 None 
                 None 
                 Mild 
               
               
                 Delay 
               
               
                 Seizures 
                 None 
                 None 
                 None 
               
               
                 Skeletal Muscle 
                 Hypotonia, 
                 Mild atrophy 
                 Weakness, Fatigue 
               
               
                   
                 Weakness, Atrophy 
               
               
                 Cardiac Muscle 
                 Dilated 
                 Dilated 
                 Dilated 
               
               
                   
                 cardiomyopathy 
                 cardiomyopathy 
                 cardiomyopathy 
               
               
                 Gastrointestinal 
                 Chronic diarrhea 
                 Normal 
                 Normal 
               
               
                 Other Clinical 
                 Pancytopenia 
                 None 
                 Hydrops, 
               
               
                   
                   
                   
                 Congestive failure 
               
               
                 Clinical Course 
                 Improved 
                 Improved 
                 Improved 
               
               
                 Highest Lactate 
                 4.8 mM 
                 2.0 mM 
                 2.5 mM 
               
               
                 Urine Organic 
                 Ketones 
                 Normal once 
                 Krebs cycle, 
               
               
                 Acids 
                   
                   
                 Generalized organic 
               
               
                   
                   
                   
                 aciduria 
               
               
                 Cranial Imaging 
                 Not done 
                 Not done 
                 CT: 
               
               
                   
                   
                   
                 Mild atrophy 
               
               
                 Muscle Biopsy 
                 Increased variation in 
                 Not done 
                 High citrate 
               
               
                   
                 muscle fiber sizes, 
                   
                 synthase 
               
               
                   
                 Increased lipid, 
               
               
                   
                 High citrate synthase, 
               
               
                   
                 Low complex 1, 3, 4 
               
               
                 Family History 
                 Maternal history of 
                 Sporadic 
                 Sporadic 
               
               
                   
                 migraine 
               
               
                   
               
               
                   
                 Patient #4 
                 Patient #5 
                 Patient #8 
               
               
                   
               
               
                 Brief Clinical 
                 Fasting 
                 Fasting 
                 Migraine/ 
               
               
                 Description 
                 hypoglycemia 
                 hypoglycemia 
                 dysautonomia 
               
               
                 Age/Sex/Race 
                 3/M/C 
                 4/F/C 
                 12/F/C 
               
               
                 Heteroplasmy 
                 16481C-T 
                 Insufficient DNA 
                 16176C-T 
               
               
                   
                   
                   
                 16338A-G 
               
               
                 Growth 
                 Normal 
                 Normal 
                 Normal 
               
               
                 Developmental 
                 None 
                 None 
                 None/gifted 
               
               
                 Delay 
               
               
                 Seizures 
                 Tonic and absence 
                 None 
                 None 
               
               
                 Skeletal Muscle 
                 Episodes of weakness 
                 Normal 
                 Normal 
               
               
                   
                 and muscle cramps, 
               
               
                   
                 Hypotonia in infancy 
               
               
                 Cardiac Muscle 
                 Normal 
                 Normal 
                 SVT 
               
               
                 Gastrointestinal 
                 Normal 
                 GER, constipation, 
                 Cyclic vomiting 
               
               
                   
                   
                 multiple episodes 
               
               
                   
                   
                 of acute 
               
               
                   
                   
                 abdominal pain 
               
               
                 Other Clinical 
                 3 episodes of 
                 Monitored for 
                 Dilated left ureter, 
               
               
                   
                 hypoglycemia 
                 possible sleep 
                 Leg cramps with 
               
               
                   
                 and/or altered 
                 apnea, 
                 exercise 
               
               
                   
                 mental status 
                 Hypoketotic 
               
               
                   
                 with fasting, 
                 hypoglycemia 
               
               
                   
                 Chronic fatigue, 
                 with fasting, 
               
               
                   
                 asthma 
                 ADHD 
               
               
                 Clinical Course 
                 Improved 
                 Improved, except 
                 Improved 
               
               
                   
                   
                 for ADHD 
               
               
                 Highest Lactate 
                 3.5 mM 
                 Normal once 
                 7.4 mM 
               
               
                 Urine Organic 
                 Krebs cycle 
                 Lactate, Ketones, 
                 Lactate, Ketones, 
               
               
                 Acids 
                   
                 Free fatty acids, 
                 Krebs cycle 
               
               
                   
                   
                 Glutarate 
               
               
                 Cranial Imaging 
                 MRI: Increased T2 
                 Not done 
                 Not done 
               
               
                   
                 signal in basal 
               
               
                   
                 ganglia 
               
               
                 Muscle Biopsy 
                 Not done 
                 Not done 
                 Increased variation 
               
               
                   
                   
                   
                 in muscle fiber 
               
               
                   
                   
                   
                 sizes, 
               
               
                   
                   
                   
                 Increased lipid, 
               
               
                   
                   
                   
                 High citrate 
               
               
                   
                   
                   
                 synthase, 
               
               
                   
                   
                   
                 Low complex 1 
               
               
                 Family History 
                 Mildly affected 
                 Unknown 
                 Maternal 
               
               
                   
                 brother 
                 (adopted) 
               
               
                   
               
               
                   
                 Patient #9 
                 Patient #10 
                 Patient #11 
               
               
                   
               
               
                 Brief Clinical 
                 Mental retardation/ 
                 Neonatal severe 
                 Mental retardation/ 
               
               
                 Description 
                 spasticity 
                 multi-system 
                 spasticity 
               
               
                   
                   
                 disease 
               
               
                 Age/Sex/Race 
                 5/F/H 
                 0/F/C 
                 5/M/H 
               
               
                 Heteroplasmy 
                 16259C-T 
                 16186C-T 
                 HV2 
               
               
                   
                 16278C-T (mom) 
               
               
                   
                 16288T-C 
               
               
                 Growth 
                 Mild short stature 
                 Failure-to-thrive 
                 Short stature, 
               
               
                   
                   
                   
                 Microcephaly 
               
               
                 Developmental 
                 Profound 
                 Vegetative 
                 Profound 
               
               
                 Delay 
               
               
                 Seizures 
                 Probable as infant 
                 Myoclonic 
                 Brief clonic 
               
               
                 Skeletal Muscle 
                 Spastic 
                 Hypertonia 
                 Spastic 
               
               
                   
                 quadriplegia 
                   
                 quadriplegia 
               
               
                 Cardiac Muscle 
                 Normal 
                 Hypertrophic 
                 Normal 
               
               
                   
                   
                 cardiomyopathy, 
               
               
                   
                   
                 Cardiac arrests 
               
               
                 Gastrointestinal 
                 Normal 
                 Esophageal atresia, 
                 Severe GER, 
               
               
                   
                   
                 Severe dysmotility 
                 Upper GI bleeding 
               
               
                 Other Clinical 
                 Optic atrophy, 
                 Acute renal 
                 Optic atrophy, 
               
               
                   
                 Strabismus, 
                 failure, 
                 Cortical blindness 
               
               
                   
                 Nystagmus 
                 Multiple 
                 Obstructive sleep 
               
               
                   
                   
                 infections, 
                 apnea 
               
               
                   
                   
                 Left superior vena 
               
               
                   
                   
                 cava and aortic 
               
               
                   
                   
                 arch 
               
               
                 Clinical Course 
                 Static 
                 Improved, then 
                 Static 
               
               
                   
                   
                 died 
               
               
                 Highest Lactate 
                 3.5 mM 
                 19 mM 
                 3.6 mM 
               
               
                 Urine Organic 
                 Normal once 
                 Lactate, Ketones, 
                 Mild ketones once 
               
               
                 Acids 
                   
                 Ethylmalonate, 
               
               
                   
                   
                 Krebs cycle, 
               
               
                   
                   
                 Generalized 
               
               
                   
                   
                 organic 
               
               
                   
                   
                 aciduria 
               
               
                 Cranial Imaging 
                 MRI: 
                 CT: 
                 MRI: 
               
               
                   
                 Leukodystrophy 
                 Thalamic infarcts 
                 Ulegyria 
               
               
                 Muscle Biopsy 
                 Not done 
                 Increased variation 
                 Focal increased 
               
               
                   
                   
                 in muscle fiber 
                 mitochondrial 
               
               
                   
                   
                 sizes, 
                 staining 
               
               
                   
                   
                 Low complex 1, 4, 
               
               
                   
                   
                 [High citrate 
               
               
                   
                   
                 synthase in 
               
               
                   
                   
                 sibling] 
               
               
                 Family History 
                 Sporadic 
                 Identically 
                 Identically affected 
               
               
                   
                   
                 affected male and 
                 male and female 
               
               
                   
                   
                 female siblings 
                 siblings who died, 
               
               
                   
                   
                 who died, 
                 First cousin parents 
               
               
                   
                   
                 HV1 heteroplasmy 
               
               
                   
                   
                 in father 
               
               
                   
               
               
                   
                 Patient #12 
                 Patient #13 
               
               
                   
               
               
                 Brief Clinical 
                 Reversible multi-system 
                 Reversible multi-system 
               
               
                 Description 
                 disease 
                 disease 
               
               
                 Age/Sex/Race 
                 3/F/H 
                 5/F/H 
               
               
                 Heteroplasmy 
                 16215A-T 
                 16285C-T 
               
               
                 Growth 
                 Failure-to-thrive, 
                 Failure-to-thrive, 
               
               
                   
                 Short stature 
                 Short stature 
               
               
                 Developmental 
                 Mild 
                 Mild-moderate 
               
               
                 Delay 
               
               
                 Seizures 
                 Complex partial 
                 In infancy only 
               
               
                 Skeletal Muscle 
                 Hypotonia 
                 Normal 
               
               
                 Cardiac Muscle 
                 Normal 
                 Normal 
               
               
                 Gastrointestinal 
                 Pyloric stenosis, 
                 Gastrostomy 
               
               
                   
                 GER, DGE 
               
               
                 Other Clinical 
                 Cleft palate 
                 Bronchopulmonary 
               
               
                   
                   
                 dysplasia, 
               
               
                   
                   
                 Ventilator dependent 
               
               
                 Clinical Course 
                 Improved 
                 Improved 
               
               
                 Highest Lactate 
                 3.4 mM 
                 5.4 mM 
               
               
                 Urine Organic 
                 Lactate, Ketones, 
                 Ketones, 
               
               
                 Acids 
                 Krebs cycle 
                 Free fatty acids, 
               
               
                   
                   
                 Generalized organic 
               
               
                   
                   
                 aciduria 
               
               
                 Cranial Imaging 
                 MRI: Normal 
                 Not done 
               
               
                 Muscle Biopsy 
                 Increased variation in 
                 Not done 
               
               
                   
                 muscle fiber sizes, 
               
               
                   
                 Increased lipid, 
               
               
                   
                 High citrate synthase 
               
               
                 Family History 
                 Maternal 
                 Maternal 
               
               
                   
               
               
                 H = Hispanic (of any race), B = Black/African American, C = Caucasian. Mixed racial children are listed as per their matrilineal race. The racial distribution in our cases resembles that of our referred patient population. The number of substitutions includes only single base pair changes, either homoplasmic or heteroplasmic, and scored as the number found in the patient/the number found in the paired haplogroup-matched control.  
               
               
                 SVT = supraventricular tachycardia, GER = gastroesophageal reflux, DGE = delayed gastric emptying, ADHD= attention deficit hyperactivity disorder. Lactate measurements listed are in plasma; normnal range 0.5-2.2 mM. Urine organic acids were performed by GC/MS; selected elevated species are listed. Kreb cycle = Kreb cycle intermediates, including fumarate, malate, and succinate.  
               
               
                 As is common in patients with other mtDNA disorders, in many of our cases normal lactates and organic acids were obtained at times while the child was clinically stable. Normal once = test performed only once and while the child was stable. In patient 4, a plasma acylcamitine profile revealed a low acetylcamitine only.  
               
               
                 In patient 5, organic acids showed increased long chain free fatty acids, 3-hydroxy free fatty acids (saturated and unsaturated C10, 12 and 14), and dicarboxylic acids (C6, 8, &amp; 10); fatty acid oxidation disorders were essentially ruled out by normal palmitate and myristate oxidation rates (M. Bennett, Dallas, Texas), normal enzymatic activities for LCHAD, SCHAD and both long and short chain 1-3-ketoacyl-CoA thiolases,  
               
               
                 # molecular testing for the common MCAD mutation and plasma acyl carnitines (showing only a non-specific increase in medium chain species, predominantly unsaturated). Muscle biopsies were performed on quadriceps; selected abnormalities are listed. Citrate synthase is a reference enzyme which when elevated can indicate mitochondrial proliferation.  
               
               
                 Low complex 1, 3, and/or 4 refer to abnormal low function of these complexes of the respiratory chain (20-50% of normal activity in each case, except for patient 6). Patient 6 received 3 biopsies at different centers by his parents&#39; request; complex 1 deficiency (1/10th of the 5th % ile) was found by Dr. John Shoffner, Scottish Rite Hospital, Atlanta, while a partial complex 4 deficiency was found by Dr. Richard Haas, University of Califomia San Diego, both on fresh muscle.  
               
               
                 As complexes 1, 3 and 4 contain mtDNA-encoded subunits, partial deficiency of each is consistent with a mtDNA mutation. Complex 5 (also containing mtDNA-encoded subunits) was not measured.  
               
             
          
         
       
     
     Patient 1 [Transient severe cardiomyopathy]. This child had an unremarkable history until at age 8 months a spider bite became infected which was followed by a loss of developmental milestones. At age 1.5 years an apparent viral upper respiratory and gastrointestinal illness with fasting and dehydration progressed to metabolic acidosis, pancytopenia and dilated cardiomyopathy with congestive heart failure. Developmental delay and failure-to-thrive (FTT) were noted at that time. A mitochondrial disorder was suspected and the child received symptomatic treatment and fasting avoidance. All clinical problems resolved within the next few months except for cyclic neutropenia, which resolved before his third birthday. Currently, at age 7 the child is clinically completely normal, including school performance and cardiac function, except for stable and symmetrical growth retardation (height age=4.7 years). The pedigree is shown in FIG.  1 . 
     Patient 2 [Transient severe cardiomyopathy]. This child presented at age 6 months with severe dilated cardiomyopathy in congestive heart failure. She was medically treated and listed for transplantation, however, her cardiac function markedly improved on strict fasting avoidance and standard medical therapy. At age 4.5 years she has near normal systolic function and possible mild decreased diastolic function. Mild failure-to-thrive also resolved. Intelligence and physical examination are normal except for a decreased muscle bulk. The family history is unremarkable. 
     Patient 4 [Fasting hypoglycemia]. At the age of 13 months, following 10 days of upper respiratory tract symptoms with decreased oral intake, this child was found to be hypoventilating, gray, staring and limp, progressing rapidly to posturing and coma. Temperature was 34.9° C. and serum glucose was 2.2 mM (40 mg/dl) when paramedics arrived; all symptoms resolved immediately following the administration of a glucose-containing intravenous fluid. A very similar event occurred following an eight-hour overnight fast. A disorder of fatty acid oxidation was postulated and fasting precautions were initiated, including 3-4 hours feedings by day and continuous drip feedings by night. No further episodes have occurred, although the child has not been fasted. Gross motor delay, mild hypotonia and growth retardation resolved. At present, the child is asymptomatic except for asthma, occasional staring spells and rare episodes of profound muscle weakness and flank/leg pain during which the child is unable to bear weight for about 2 hours. Family history is remarkable only for a cystic hygroma and similar episodes of muscle weakness, generalized fatigue and an inability to walk in his now 5 year old brother. Their normal parents are not consanguineous. 
     Patient 9 [Mental retardation/spasticity]. This child has a static encephalopathy with profound mental retardation, spastic quadriplegia, strabismus and nystagmus. Signs or symptoms of non-neuromuscular tissue dysfunction have been absent. Diagnostic evaluation revealed optic atrophy and leukodystrophy. The family history is unremarkable. 
     Patient 10 [Neonatal severe multi-system disease]. This child presented at birth with esophageal atresia and a severe metabolic acidosis refractory to standard treatment. He was transported to the Childrens Hospital Los Angeles facility in severe cardiogenic shock at age 11 days and started on ionotropes for a hypertrophic cardiomyopathy, peritoneal dialysis for acute renal failure and specific therapy for mitochondrial failure. The latter was designed in order to reverse and prevent catabolism and consisted of a continuous infusion of glucose at 10-12 mg/kg/min, insulin at about 0.1 units/kg/hr (titrated to maintain serum glucose between 5-10 mM=90-180 mg/dl), and protein at 1.0 g/kg/d. On this treatment, the anion gap (30 to 14 mM), plasma lactate and renal function normalized, and cardiac function greatly improved within one week. Of note, the lactate remained elevated for several days following the return of normal circulation. The child remained ventilator dependent with chronic lung disease and suffered several cardiac arrests; the last one being lethal at age 4 months. The family history is remarkable for a previous male and female sibling with very similar manifestations (including organic acids and muscle electron transport chain activities in one), who died at ages 3 and 5 months despite intensive support. 
     Patient 12 [Reversible multi-system disease]. This child was delivered at 28 weeks gestation following a pregnancy reportably complicated by cocaine and ethanol exposure, although facial morphology appears normal. However, except for chronic lung disease and brief mixed apnea, there were no other complications related to prematurity. The child&#39;s first year was dominated by severe symmetrical FTT and gastrointestinal dysfunction; a fundoplication/gastrostomy and pyloroplasty were performed for gastroesophageal reflux (GER) and pyloric stenosis. Additional problems included complex partial seizures, hypotonia and cleft palate. All of these conditions either resolved or were surgically repaired, and presently at age 3 years her only problem is mild global cognitive delay. Although she remains very small (height and weight age=17 and 13 months, respectively), she now demonstrates a normal growth rate for age. The child was adopted, although her 4 year old maternal half-sister was evaluated and found to have symmetrical FTT with a normal current growth rate, moderate developmental delay, strabismus, and resolved GER. 
     EXAMPLE 3 
     Methods 
     Subjects. The experimental group consisted of 67 unrelated children ascertained over a five year period (1994-1999) from the clinical genetics practice of Dr. Richard Boles at Childrens Hospital Los Angeles. All qualifying children were retrospectively recruited based upon the presence of neuromuscular and/or multi-system system disease, an elevated body fluid lactate concentration (usually in plasma and often minimally elevated, &gt;2.0 mM or 18 mg/dl) and the absence of another diagnosis despite an extensive evaluation. This latter evaluation was tailored to each specific child, but always included a high resolution karyotype, urine organic acids, plasma amino acids, Southern blotting for large rearrangements, and PCR for 10 known point mutations (Wong and Senadheera (1997)  Clin Chem  43:1857-61). An additional 14 children that would have qualified were excluded from the study due to the absence of available DNA from a blood sample. No cases were included if ascertainment was based on the referral of a sample to the laboratory for the purpose of mtDNA analysis. 
     The control group consisted of 103 unrelated individuals, of which 76 were children of all ages, with definitive diagnoses of non-mitochondrial disorders, including 58 individuals with phenylketonuria (PKU) diagnosed on newborn screening, and 33 and 12 individuals diagnosed by molecular assays with spinal muscular atrophy and Duchenne muscular dystrophy, respectively. There was no reason to believe that any of these conditions were related to mtDNA sequence variations. This study was approved by the CHLA Institutional Review Board. 
     Molecular Assays. Total DNA was extracted from blood or hair root and PCR and temporal temperature gradient gel electrophoresis (TTGE) were performed as previously reported (Zoller and Redilla-Flores (1996) Temporal temperature gradient gel electrophoresis of cystic fibrosis samples on the Dcode system. Bio-Rad Laboratories, US/EG Bulletin 2103; Chen et al. (1999)  Clin Chem  45:1162-7; Higashimoto et al. (1999)  Clin Chem  45:2005-6; Wong and Lam (1997)  Clin Chem  43:1241-3). Peripheral blood was assayed in all cases and controls. When available, hair was assayed in the probands and blood and/or hair was assayed in their first degree relatives. The mtDNA control region was divided into 3 segments for TTGE analysis (FIG.  2 ). TTGE was a relatively novel heteroduplex detection assay which has been shown to be very sensitive in the detection of mtDNA heteroplasmy (including 60 of 60 known cases positively identified (Chen et al. (1999)  Clin Chem  45:1162-7; Chen et al. (1997)  Am J Hum Genet  61:A306 (abstract)). TTGE was also very specific as each heteroplasmic mutation or polymorphism demonstrated a reproducible and distinct band pattern (FIG. 3) (Chen et al. (1999)  Clin Chem  45:1162-7). Heteroplasmy could be detected with proportions as low as about 5% (supra). Ethidium bromide stained TTGE gels were visualized under UV light and imaged with a digital CCD gel documentation system. Percent heteroplasmy for each sequence variant could readily be estimated by the relative intensities of the bands. These measurements could be taken by eye or Gene scan analysis with fluorescent labeled primers and employing the binomial theorem, with results closely agreeing those obtained by denaturing high performance liquid chromatography (Transgenomic WAVE™ DNA Fragment Analysis System, Omaha, Nebr., data not shown). 
     For quality assurance, every gel was run concurrently with 1-3 positive controls (known heteroplasmic samples). All assays were repeated starting with the original extracted DNA sample in every case in which heteroplasmy was found in order to exclude PCR artifacts. Pseudogenes were not amplified in any of the segments as determined by the absence of any PCR product (FIG. 3) using primers listed in Table 2 with total DNA extracted from rho negative cell culture (cells lacking any mtDNA (Chomyn et al. (1991)  Mol Cell Biol  11:2236-44), kindly provided by Anne Chomyn at CalTech). Contaminated (i.e. blood transfused) samples were easily detected by the presence of multiple bands (&gt;4) present in multiple mtDNA segments. One such sample was thus identified and removed from this study after confirmation of contamination by HLA typing. Cyclosequencing was performed in all cases with multiple bands seen on TTGE either from a single band cut out from the gel or directly from the original samples, using a dye terminator Cyclosequencing kit (Applied Biosystems) and an Applied Biosystems ABI 373A DNA Sequencer. Haplogrouping was determined by restriction digest and sequencing part of the control region as previously reported (Torroni et al. (1996)  Genetics  144:1835-50). 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Segment 
                 mtDNA Regions 
                 Nucleotides 
                 Size (bp) 
                 Primer Sequences (5′-3′) 
                 Ta 
                 Ti-Tf, Tr 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 7S 
                 Control region 
                 15987-16509 
                 523 
                 upper 
                 CACCCAAAGCT 
                 53 
                 50-62, 3.0 
               
               
                   
                 (includes HV1); 
                   
                   
                   
                 AAGATTCTAA 
               
               
                   
                 tRNA-Pro 
                   
                   
                   
                 (SEQ ID NO:1) 
               
               
                   
                   
                   
                   
                 lower 
                 AGGCTTTATGAC 
               
               
                   
                   
                   
                   
                   
                 CCTGAAGTA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:2) 
               
               
                 7Sb* 
                 Control region 
                 16200-16509 
                 310 
                 upper 
                 TCCACATCAAAA 
                 53 
                 56-64, 3.0 
               
               
                   
                   
                   
                   
                   
                 CCCCCCCCC 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:3) 
               
               
                   
                   
                   
                   
                 lower 
                 AGGCTTTATGAC 
               
               
                   
                   
                   
                   
                   
                 CCTGAAGTA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:4) 
               
               
                 7Sc* 
                 Control region 
                 15852-16155 
                 304 
                 upper 
                 TCTCCCTAATTG 
                 58 
                 50-59, 3.0 
               
               
                   
                   
                   
                   
                   
                 AAAACAAAA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:5) 
               
               
                   
                   
                   
                   
                 lower 
                 TGGATTGGGTTT 
               
               
                   
                   
                   
                   
                   
                 TTATGTACT 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:6) 
               
               
                 CSB 
                 Control region 
                 16407-560  
                 723 
                 upper 
                 CCTCCGTGAAAT 
                 53 
                 54-61, 3.7 
               
               
                   
                 (includes HV2) 
                   
                   
                   
                 CAATATCCC 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:7) 
               
               
                   
                   
                   
                   
                 lower 
                 AAACTGTGGGG 
               
               
                   
                   
                   
                   
                   
                 GGTGTCTTTG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:8) 
               
               
                 F 
                 Control region 
                  500-1190 
                 691 
                 upper 
                 CCCATCCTACCC 
                 56 
                 55-59, 1.0 
               
               
                   
                 tRNA-Phe; 
                   
                   
                   
                 AGCACACAC 
               
               
                   
                 12S-rRNA 
                   
                   
                   
                 (SEQ ID NO:9) 
               
               
                   
                   
                   
                   
                 lower 
                 GATATGAAGCA 
               
               
                   
                   
                   
                   
                   
                 CCGCCAGGTC 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:10) 
               
               
                   
               
               
                 TTGE conditions for each segment were determined by computer simulation (MacMelt, Bio-Rad Laboratories) and adjusted by experimentation. T a  is the annealing temperature for PCR. T 1 , T f , and T r  are the initial, final, and ramp rate temperatures for TTGE.  
               
               
                 *A common length heteroplasmy at nt 16184 in the 7S segment occasionally complicated the search for potential additional point heteroplasmies elsewhere in the same segment. These cases were each evaluated by TTGE in two additional segments ‘7Sb’ and ‘7Sc’ which excluded the site of the length heteroplasmy by moving one of the primer sites to a conserved sequence area close to the homopolymeric cytosine tract.  
               
             
          
         
       
     
     EXAMPLE 4 
     Results 
     Distinct TTGE band patterns consistent with the presence of a heteroplasmic single nucleotide substitution (‘point heteroplasmy’, FIG. 3) were found in the 7S fragment in 10 of 67 children in the experimental group and in none of the 103 individuals in the control group (p&lt;0.001). In the 8/10 cases in which sufficient DNA was available, sequencing confirmed the presence of at least one point heteroplasmy in hypervariable region 1 (HV1, Table 1, FIG. 2; one mutation was outside of the region commonly referred to as HV1). TTGE analysis in the CSB fragment was consistent with point heteroplasmy in 1 case and in 0 controls; and sequencing revealed a heteroplasmic nucleotide in HV2 (Table 1, FIG.  2 ). In each case, these base substitutions were previously identified in homoplasmic form as polymorphisms (found in normal individuals) (Kogelnik et al. (1996)  Nuc Ad Res,  24:177-9; Opdal et al. (1998)  Acta Paediatr  87:1030-44), and many of these polymorphisms have been encountered upon sequencing control individuals in the laboratory (data not shown). Elsewhere in the mtDNA control region, point heteroplasmy was absent in all patients and controls. Multiple cases in both the experimental and control groups were revealed by TTGE and confirmed by sequencing to have polymorphic length variants (length heteroplasmy). Most of these length variants have been previously reported (Bendall and Sykes (1995)  Am J Hum Genet  57:248-56; Torroni et al. (1994)  Am J Hum Genet  55:760-76), although rare or unique variants were also found in at least 2 patients and are the subject of continuing investigation. 
     In order to determine whether the observed point heteroplasmic variants were inherited or somatic mutations, samples were assayed from the family members of patients. Consistent with maternal inheritance of mtDNA, in each case in which samples from the mother (patients 4, 6-10, 13), full sibling (patients 4, 6, 9, 10, 13) or maternal half sibling (patient 12) were available, the identical point heteroplasmy was identified by TTGE and/or sequencing. Samples were available from the fathers of patients 1, 4, 6, 7, 9 and 10 and the specific nucleotide variation seen in the proband was absent in each. A distinct HV1 point heteroplasmy was noted in the father of patient 10. DNA was not available from any family members of the child with HV2 heteroplasmy. 
     Direct sequencing of the patient or family samples rarely reveals heteroplasmy due to low proportions of the minority sequence (often about 5-25% as estimated by TTGE). The methods employed herein, which involve sequencing of DNA derived from different gel bands, readily reveals the two sequences constituting the heteroplasmic nucleotide identified by TTGE, but this does not exclude the presence of other heteroplasmic nucleotides within the same segment. Careful review of the HV1 sequences revealed probable point heteroplasmy in additional nucleotides in many cases, but not in controls. A second heteroplasmic nucleotide, suspected by sequencing, was confirmed using PCR/allele specific oligonucleotide (ASO) analysis (Wong and Senadheera (1997)  Clin Chem  43:1857-61) in patients 8 and 9 (Table 1). In the latter, further PCR/ASO analyses in the asymptomatic (by history and neurological examination) mother and 11 year old sister revealed the presence of point heteroplasmy at both nucleotides in the mother, and at one site only in the sister (16288T-C). In addition, a third point heteroplasmy was found in the mother, which was homoplasmic for the polymorphism (16278T) in our patient by PCR/ASO. At each heteroplasmic loci, the proportions of each variant were widely different in each family member (and in individual hair roots within each individual), such that almost all direct sequences revealed homoplasmy (single mtDNA sequence present) for one or the other sequence variant. 
     In conclusion, reported herein is a novel association of predominately intermittent and non-progressive disease, biochemical signs suggestive of mitochondrial dysfunction, and maternally-inherited mtDNA-CR point heteroplasmy. Pathology is likely mediated through a novel disease mechanism. This disorder is apparently not rare as it was identified in 1/6 children with idiopathic neuromuscular or multi-system disease and an elevated body fluid lactate, a paradigm which is frequently seen in tertiary practice. With highly variable manifestations, this condition(s) will likely be encountered by clinicians in a variety of specialties treating children. Recognition is important, not only to provide the family with a specific diagnosis that in cases without severe neuromuscular disease appears to carry a relatively favorable prognosis, but in many cases to provide appropriate treatment, especially regarding the avoidance of fasting. 
     The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. 
     
       
         
               
             
           
               
                   
               
             
             
               
                                    
               
               
                 #             SEQUENCE LISTING  
               
               
                   
               
               
                   
               
               
                 &lt;160&gt; NUMBER OF SEQ ID NOS:   10  
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 1  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 1  
               
               
                   
               
               
                 cacccaaagc taagattcta a            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 2  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 2  
               
               
                   
               
               
                 aggctttatg accctgaagt a            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 3  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 3  
               
               
                   
               
               
                 tccacatcaa aacccccccc c            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 4  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 4  
               
               
                   
               
               
                 aggctttatg accctgaagt a            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 5  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 5  
               
               
                   
               
               
                 tctccctaat tgaaaacaaa a            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 6  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 6  
               
               
                   
               
               
                 tggattgggt ttttatgtac t            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 7  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 7  
               
               
                   
               
               
                 cctccgtgaa atcaatatcc c            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 8  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 8  
               
               
                   
               
               
                 aaactgtggg gggtgtcttt g            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 9  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 9  
               
               
                   
               
               
                 cccatcctac ccagcacaca c            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 10  
               
               
                 &lt;211&gt; LENGTH: 21  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: synthetic construct  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 10  
               
               
                   
               
               
                 gatatgaagc accgccaggt c            
               
               
                 #                   
               
               
                 #                   
               
               
                 #21