All In One Application Note - Savyon Diagnostics€¦ · Israeli population comprised of many...

02-05.2016 AP800014E

All In One Application Note

Test kit for 21 samples REF: 800014 Store at -20°C For use with the NanoChip® 400 Instrument

For Professional Use Only

Savyon Diagnostics Ltd. 3 Habosem St. Ashdod 7761003 ISRAEL Tel.: +(972).8.8562920 Fax: +(972).8.8523176 E-mail:

[email protected]

European Authorized Representative: Obelis s.a. Boulevard Général Wahis 53 1030 Brussels, BELGIUM Tel: +(32) 2. 732.59.54 Fax: +(32) 2.732.60.03

E-Mail : [email protected]

mailto:[email protected]:[email protected]

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Table of Contents

Introduction ................................................................................................. 3 Intended Use ................................................................................................ 3 Background .................................................................................................. 3 Kit Contents ................................................................................................. 12 Storage ...................................................................................................... 12 Using NanoChip Cartridges .............................................................................. 12

NanoChip Cartridge Handling ...................................................................... 12

Materials and Equipment ................................................................................. 13 Materials Available from................................................................................... 13 Additional Materials Available from Savyon ............................................................ 13 Other Required Materials (not available from Savyon ) ............................................... 14 Required Equipment ....................................................................................... 14

Technical Assistance ...................................................................................... 14 Precautions ................................................................................................. 15 Performing Sample Amplification ....................................................................... 16 Extraction ................................................................................................... 16 Amplification ................................................................................................ 16 Preparing the Sample Plate .............................................................................. 19

Operating the NanoChip 400 System .................................................................... 20 Preparing Solutions for Use in the NanoChip 400 Instrument ....................................... 21 Preparing the NanoChip Cartridge and Instrument .................................................... 21 Creating a Protocol ........................................................................................ 22 Running the Assay ......................................................................................... 25 Analyzing the Data ......................................................................................... 27

Appendix A: All In One Assay Format .................................................................. 28 Appendix B: All In One Data Analysis Spreadsheet and Data Calculations ....................... 34 Appendix C: Legal Notices ............................................................................... 37 REFERENCES ................................................................................................ 38

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Introduction

Intended Use

The NanoChip® All In One Kit is used to detect and identify a panel of 35 different genetic diseases (91

mutations) that are associated with various genetic disorders of the Israeli population, based on the specific individual ethnic origin.

For in vitro diagnostic use only.

Background

Israeli population comprised of many ethnic backgrounds. Different ethnic groups have a higher risk for specific disease-causing mutations than the general Israeli population. These diseases are inherited in an autosomal recessive pattern. Affected individuals have inherited two copies of the mutated gene, one from each parent. The All In One assay was designed to diagnose the following diseases:

3 Methyl Glutaconic Aciduria (3MGA), also known as Costeff Optical Atrophy, is characterized by degeneration (atrophy) of the optic nerves, which leads to visual acuity, within the first years of life. Other nervous system problems might occur, such as an inability to maintain posture, poor muscle tone, a gradual increase of involuntary jerking movements (choreiform movements), and a general decrease in brain function (cognitive deficit). The disorder is caused by mutations in the OPA3 gene encoding a protein whose function is unknown. Researchers have suggested that cells with a defective OPA3 protein are prematurely vulnerable to apoptosis. The incidence of 3-methylglutaconic aciduria type III is about 1 in 10,000 newborns in the Iraqi Jewish population. This disorder is extremely rare in all other populations. Carrier frequency in affected populations is estimated in 1 in 10 to 40 in Iraqi Jews and in 1 in 10 in Persian (Iranian) Jews (1-2). Alpha 1- Antitrypsin (AAT) deficiency is the most prevalent potentially lethal hereditary disease of Caucasians. It leads to jaundice in infants, liver disease in children and adults, and pulmonary emphysema in adults. AAT is a protease inhibitor (PI), which protects tissue structures from damage by degrading enzymes. The genetic defect in AAT deficiency results in a molecule that cannot be released from its production site in hepatocytes. Low serum levels of AAT result in low alveolar concentration, where the protein normally would serve as protection against proteases. Consequential protease excess destroys alveolar walls and causes obstructive lung disease. Moreover, unsecreted AAT self-aggregates in the liver and causes liver disease. Mutations in the PI locus, located on chromosome 14, are associated with AAT deficiency. The most common risk alleles are PiS whose worldwide carrier rate is 1:50 (1:9 to 1:12 in Caucasians) and PiZ, with a worldwide carrier rate of 1:162 (1:30 to 1:40 in Caucasians) (3-8).

Ataxia Telangiectasia (AT), is an autosomal recessive disorder characterized by progressive difficulty with coordinating movements (ataxia) beginning in early childhood, usually before age 5. Affected children typically develop difficulty walking, problems with balance and hand coordination, involuntary jerking movements (chorea), muscle twitches (myoclonus), and disturbances in nerve function (neuropathy). The movement problems typically cause people to require wheelchair assistance by adolescence. People with this disorder also have slurred speech and trouble moving their eyes to look side-to-side (oculomotor apraxia). Small clusters of enlarged blood vessels called telangiectases, which occur in the eyes and on the surface of the skin, are also characteristic of this condition. People with ataxia-telangiectasia often have a weakened immune system, and many develop chronic lung infections. They also have an increased risk of developing cancer, particularly cancer of blood-forming cells (leukemia) and cancer of immune system cells (lymphoma). Affected individuals are very sensitive to the effects of

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radiation exposure, including medical x-rays. The life expectancy of people with ataxia-telangiectasia varies greatly, but affected individuals typically live into early adulthood. Complementation groups for the classic form of the disease map to chromosome 11q23 and are associated with mutations in the ATM gene. Ataxia-telangiectasia occurs in 1 in 40,000 to 100,000 people worldwide. Carrier frequency is particularly high among North African Jews estimated at 1 in 40 to 80 individuals (9). Bloom Syndrome is inherited in an autosomal recessive fashion. Bloom Syndrome patients are much smaller than average, and often have a high-pitched voice and characteristic facial features including a long, narrow face, small lower jaw, a prominent nose and ears. They tend to develop pigmentation changes and dilated blood vessels in the skin. Other features of the disorder may include learning disabilities, mental retardation, chronic lung problems and diabetes. Men with Bloom Syndrome usually do not produce sperm while women with the disorder generally experience menopause earlier than usual. Chromosomal instability in Bloom Syndrome results in a high risk of cancer in affected individuals. Mutations in the BLM gene (locus 15q26.1) cause Bloom Syndrome. The BLM gene provides instructions for producing a protein called the Bloom (BLM) Syndrome Protein, which is a member of the DNA helicase family The carrier frequency in individuals of Eastern European ancestry is about 1:100 (10-11). Canavan Disease is an autosomal recessive disorder that causes progressive damage to nerve cells in the brain. This disease is one of a group of genetic disorders called leukodystrophies. They are characterized by degeneration of myelin in the phospholipid layer insulating the axon of a neuron. The gene is located on chromosome 17. Canavan disease is caused by a defective ASPA gene which is responsible for the production of the enzyme aspartoacylase. Decreased aspartoacylase activity prevents the normal breakdown of N-acetyl aspartate, and the lack of breakdown somehow interferes with growth of the myelin sheath of the nerve fibers in the brain. Although Canavan disease may occur in any ethnic group, it affects persons of Eastern European Jewish ancestry more frequently. About 1/40 individuals of Ashkenazi Jewish ancestry are carriers. (12-14).

Cystic fibrosis (CF) is a common recessive disease caused by mutations in the CFTR gene (18). The hallmark symptoms of cystic fibrosis are: salty tasting skin,(15) poor growth and poor weight gain despite a normal food intake,(16) accumulation of thick, sticky mucus, frequent chest infections and coughing or shortness of breath.(17) However the disease phenotype varies from one patient to another. Several mutations primarily the 5T, is known to generate Congenital Bilateral Absence of the Vas Deferens (CBAVD) which is found in otherwise healthy infertile males, CBAVD is associated with a high incidence of mutated CFTR alleles, and is considered a genital form of cystic fibrosis (CF).

Connexin or Congenital deafness occurs in 1 per every 1000-2000 births with autosomal recessive inheritance being the most common form (more than 75%). In European, North American and Mediterranean populations a common allele, designated 35delG, accounts for 3/4 or more of all GJB2 mutations (19, 20 and 21). The 167delT mutation is recurrently found in Ashkenazi Jewish populations with a probable carrier frequency of 3–4%. Mutations in GJB6, encoding for Cx30, a deletion of 342Kb involving GJB6 has been reported to cause deafness both in the homozygous status and in heterozygousity with a GJB2 point mutation in trans (22). Cerebrotendinous Xanthomatosis (CTX) is a fat (lipid) storage disorder that affects many areas of the body. People with this disorder cannot break down certain lipids effectively, specifically different forms of cholesterol, so these fats accumulate in various areas of the body. Characteristic clinical manifestations of cerebrotendinous xanthomatosis include chronic diarrhea during infancy, clouding of the lens of the eye (cataracts) developing in late childhood, progressively brittle bones that are prone to fracture, and neurological problems in adulthood, such as dementia, seizures, hallucinations, depression, and difficulty with coordinating movements (ataxia) and speech (dysarthria). The neurological symptoms are thought to be caused by an accumulation of fats and an increasing number of xanthomas (fatty yellow nodules) in the brain. Xanthomas can also accumulate in the fatty substance that

http://en.wikipedia.org/wiki/Autosomal_recessivehttp://en.wikipedia.org/wiki/Nerve_cellhttp://en.wikipedia.org/wiki/Brainhttp://en.wikipedia.org/wiki/Leukodystrophieshttp://en.wikipedia.org/wiki/Myelinhttp://en.wikipedia.org/wiki/Phospholipidhttp://en.wikipedia.org/wiki/Axonhttp://en.wikipedia.org/wiki/Neuronhttp://en.wikipedia.org/wiki/Cystic_fibrosis#cite_note-pmid17557942-6#cite_note-pmid17557942-6http://en.wikipedia.org/wiki/Cystic_fibrosis#cite_note-pmid15339250-7#cite_note-pmid15339250-7http://en.wikipedia.org/wiki/Cystic_fibrosis#cite_note-pmid19393108-9#cite_note-pmid19393108-9

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insulates and protects nerves (myelin), disrupting nerve signaling in the brain. Disorders that involve the destruction of myelin are known as leukodystrophies. Degeneration (atrophy) of brain tissue caused by excess lipid deposits also contributes to the neurological problems. Xanthomas in the tendons (most commonly in the Achilles tendon, which connects the heel of the foot to the calf muscles) begin to form in early adulthood. Tendon xanthomas may cause discomfort and interfere with tendon flexibility. People with cerebrotendinous xanthomatosis are also at an increased risk of developing cardiovascular disease. The incidence of cerebrotendinous xanthomatosis is estimated to be 3 to 5 per 100,000 people worldwide. This condition is more common in the Moroccan Jewish population with an incidence of 1 in 108 individuals. The carrier frequency among North African Jews is 1 in 50 to 80 individuals (23-24). Dihydrolipoamide dehydrogenase deficiency (DLD) also known as Lipoamide Dehydrogenase Deficiency (LDD), DLD deficiency, E3 deficiency and, Maple syrup urine disease type III, is an autosomal recessive metabolic disorder characterized biochemically by a combined deficiency of the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), pyruvate dehydrogenase complex (PDC), and alpha-ketoglutarate dehydrogenase complex (KGDC). Clinically, affected individuals have lactic acidosis and neurologic deterioration due to sensitivity of the central nervous system to defects in oxidative metabolism. LDD is often associated with increased urinary excretion of alpha-keto acids, such as pyruvate. The deficiency can also be associated with increased concentrations of branched-chain amino acids, as observed in maple syrup urine disease (MSUD), and is sometimes referred to as 'MSUD type III,' although patients with LDD have additional biochemical defects. Carrier frequency is 1:90 among the Ashkenazi Jewish population (25-28). Fanconi anemia Type A (FANCA) can be caused by homozygous or compound heterozygous mutation in the FANCA gene on chromosome 16q24.3. Mutations in this gene are the most common cause of Fanconi anemia. Fanconi anemia is a clinically and genetically heterogeneous disorder that causes genomic instability. Characteristic clinical manifestations of Fanconi anemia include pre- and postnatal growth retardation; developmental abnormalities in major organ systems (malformations of the kidneys, heart, and skeleton-absent or abnormal thumbs and radii; a typical facial appearance with small head, eyes, and mouth; hearing loss; hypogonadism and reduced fertility; cutaneous abnormalities (hyper- or hypopigmentation and cafe-au-lait spots); bone marrow failure; and high predisposition, susceptibility, to cancer, predominantly acute myeloid leukemia. The cellular hallmark of FA is hypersensitivity to DNA crosslinking agents and high frequency of chromosomal aberrations pointing to a defect in DNA repair. The life expectancy of FA patients is reduced to an average of 20 years (range, 0-50). Fanconi anemia of complementation group A is common among Moroccan Jews. The carrier frequency is 1:100 in North African Jews (29-30). Fanconi Anemia C (FANCC) is an autosomal recessive disease characterized by progressive bone marrow failure, congenital anomalies, aplastic anemia and cancer susceptibility. There are at least 8 complementation groups (A-H). Extensive analysis of the FA group C gene FANCC in western countries revealed that 10-15% of FA patients have mutations in this gene. FANCC was mapped to chromosome 9 .The most common mutation is IVS4+4 A to T, a splice mutation in intron 4 which has been found mainly in patients of Ashkenazi Jewish ancestry. Patients with IVS4 mutation have a severe phenotype in comparison to other FA patients. The carrier frequency of the defective gene is 1:80 and the disease frequency is 1:25,600 within the Ashkenazi Jewish population (31). Familial Dysautonomia (FD), also known as “Riley-Day Syndrome” or “Hereditary Sensory Neuropathy Type III”, is an autosomal recessive disorder that affects the development and survival of sensory, sympathetic, and some parasympathetic neurons. Individuals with FD are affected with a variety of symptoms, which include decreased sensitivity to pain and temperature, cardiovascular instability, recurrent pneumonias, vomiting crises, and gastrointestinal dysfunction (32, 33, 34 and 35). The major haplotype of FD is associated with mutation (2507+6T>C) that affects the donor splice site of intron 20 of the IKBKAP gene (37, 38). FD disorder is primarily confined to individuals of Ashkenazi Jewish descent. The carrier frequency of the defective gene is 1:30 and the disease frequency is 1:3600 within the Ashkenazi Jewish population (36).

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Galactosemia also known as Galactose-1-phosphate uridylyltransferase deficiency and GALT deficiency, is an autosomal recessive disorder of galactose metabolism. Most patients present in the neonatal period, after ingestion of galactose, with jaundice, hepatosplenomegaly, hepatocellular insufficiency, food intolerance, hypoglycemia, renal tubular dysfunction, muscle hypotonia, sepsis, and cataract. Long-term complications include mental retardation, verbal dyspraxia, motor abnormalities, and hypergonadotropic hypogonadism. A common mutation among Ashkenazi Jewish population is a 5.5kb deletion in the GALT gene and the carrier frequency among this population is 1:127 (39). Gaucher Type 1 (GD) is the most common lysosomal storage disorder. GD is characterized by hereditary reduced activity of the lysosomal β-glucocerebrosidase which is encoded by the GBA gene located to chromosome 1q21 (40, 42). GD is inherited in an autosomal recessive pattern. A pseudogene of GBA, located 16 Kb downstream from the gene, share 96% DNA sequence homology with the active gene (43). The carrier rate for the mutations which cause GD may be as high as 1 in 15 Jewish people of Eastern European ancestry, and 1 in 100 of the general population (41). Glycogen Storage Disease Type 1a (GSD1a/ Von Gierke Disease) is an autosomal recessive disorder with an incidence of 1 in 100,000 births. GSD type 1a is caused by deficiency in the activity of glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. Individuals with GSD1a exhibit a wide range of clinical symptoms including growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia, tendency to bleed, neutropenia, hepatic adenomas and renal failure. GSD1a is caused by mutations in the G6Pase gene, located on chromosome 17. R83C is the most common mutation among Caucasians (44-45).

Glycogen Storage Disease Type 3 (GSD3) begins to manifest in the first few months of life. Periods of fasting, of varying lengths, can trigger hypoglycemia and can lead to more severe symptoms such as seizures or respiratory distress. One of the first visible indicators of GSD III is a swollen or distended belly, which is caused by the buildup of glycogen and enlarging the liver. This can also lead to jaundice, cirrhosis and liver failure. GSD III is considered a muscular dystrophy. Poor muscle tone may be present early on, and later in life some individuals may experience decreased mobility or heart problems due to progressive weakness in the skeletal and/or cardiac muscle. Disease Frequency is estimated in 1 in 5,400 in North African Jews and 1 in 100,000 in the general population. Carrier Frequency is 1 in 35 in North African Jews (46-47).

Hereditary Inclusion Body Myopathy (HIBM), also known as inclusion body myopathy 2 (IBM2), is caused by homozygous or compound heterozygous mutation in the GNE gene. Inclusion body myopathy 2 primarily affects skeletal muscles, causes muscle weakness that appears in late adolescence or early adulthood and worsens over time. The first sign of inclusion body myopathy 2 is weakness of a muscle in the lower leg, the tibialis anterior. Weakness in this muscle alters the way a person walks and makes it difficult to run and climb stairs. As the disorder progresses, weakness also develops in muscles of the upper legs, hips, shoulders, and hands. Most people with inclusion body myopathy 2 require wheelchair assistance within 20 years after signs and symptoms appear. People with the characteristic features of inclusion body myopathy 2 have been described in several different populations. The disorder was first described in Jews of Persian descent however, it was later found also in Jews originating from other Middle Eastern countries, as well as in non-Jews. Carrier frequency among Iranian Jews is estimated in 1 in 38 individuals (48-49). Infantile Cerebral and Cerebellar Atrophy (ICCA) patients present microcephaly of postnatal onset, epilepsy, and psychomotor retardation. Head circumference percentiles decline with age, and brain MRI shows cereberal and cerebellar atrophy with severe myelination defect. Microcephaly of prenatal onset has been linked to disruption of genes that play a role in cell division, chromosome segregation, and centrosome function. Abnormal tone and seizures are common. Patients with ICCA carry the same mutation in the MED17 gene and belong to the ethnic group of Caucasus Jews. Carrier frequency among this population is 6.25% (1in 16) (50).

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Joubert Syndrome 2 (JBTS2) is a genetically heterogeneous autosomal recessive disorder characterized by psychomotor retardation, hypotonia, ataxia, nystagmus, and oculomotor apraxia and variably associated with dysmorphism, as well as retinal and renal involvement (51). JBTS2 is caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. In Ashkenazi Jewish, a single G35T mutation in exon 4 of the TMEM216 gene, results in an arg73-to-leu (R73L) substitution was identified as a founder mutation with a carrier rate of 1 in 92 (52).

Limb Girdle Muscular Dystrophy Type 2B (LGMD2b), also known as dysferlinopathy, is caused by mutations in the DYSF gene encoding the skeletal muscle protein dysferlin. The disease causes weakness and wasting of the muscles in the arms and legs. The muscles most affected are those closest to the body (proximal muscles), specifically the muscles of the shoulders, upper arms, pelvic area, and thighs. Symptoms begin in the lower legs/pelvic region with fatigue and difficulty climbing stairs and standing from a squatting position. Symptoms typically progress within 10 years to the upper body/shoulder region with difficulty raising arms above the head. The use of a cane occurs on average about 14 years after onset of symptoms and individuals require a wheelchair about 21 years after onset of symptoms. The respiratory and heart muscles are not typically affected. Intelligence is generally unaffected in limb-girdle muscular dystrophy; however, developmental delay and intellectual disability have been reported in rare forms of the disorder. The disease affects Libyan, Yemenite and Caucasus Jewish populations yet, is most common among Jews of Libyan origin. Carrier frequency is 1in 10 in this population and disease prevalence is at least 1 per 1300 adults (53-54).

Mucolipidosis (ML4) is a group of metabolic disorders inherited in an autosomal recessive manner. In ML4 patients, abnormal amounts of carbohydrates and lipids accumulate in cells. The symptoms range from mild learning disabilities to severe mental retardation and skeletal deformities. ML4 is classified as a lysosomal storage disease. The gene responsible for ML IV MCOLN1 makes the protein mucolipin-1. Due to mutations in the gene, mucolipin-1 is missing or dysfunctional in people with ML 4. The gene is mapped to a chromosomal region 19p13.2-13.3. Carrier frequency of ML4 is 1:110 and 1:40,000 births (55) Megalencephalic Vacuolating Leukoencephalopathy (MLC1) is a progressive condition that affects brain development and function and is caused by mutations in the MLC1 gene on chromosome 22q13.33. The MLC1 gene encodes a protein that is found in the brain, spleen, and white blood cells (leukocytes). Individuals with this condition typically have an enlarged brain (megalencephaly) that is evident at birth or within the first year of life. Megalencephaly progressively increases the size of the head. Affected people also have leukoencephalopathy, an abnormality of the brain's white matter (nerve fibers covered by myelin) namely, a swollen myelin stippled with vacuoles. Over time, the swelling decreases and the myelin begins to waste away (atrophy). Individuals affected with this condition may develop subcortical cysts below an area of the brain called the cerebral cortex. These cysts can grow in size and number. This condition may lead to abnormal tensing of the muscles (spasticity) and difficulty coordinating movements (ataxia). Walking ability varies greatly among those affected. Some people lose the ability to walk early in life and need wheelchair assistance, while others are able to walk unassisted well into adulthood. Affected individuals may also develop abnormal muscle tone (dystonia), involuntary writhing movements of the limbs (athetosis), difficulty swallowing (dysphagia), and impaired speech (dysarthria). People with this condition typically have only mild to moderate intellectual impairment. More than half of all people with this condition have recurrent seizures (epilepsy) which can occur without warning or may follow minor head trauma. The disease is particularly common among Jews of Libyan origin. Carrier frequency in this population is 1 in 40 individuals (56-58). Metachromatic Leukodystrophy, infantile type MLD is an inherited disorder characterized by the accumulation of fats called sulfatides in cells. This accumulation especially affects myelin producing cells in the nervous system. Sulfatide accumulation in myelin-producing cells causes progressive destruction of white matter (leukodystrophy) throughout the nervous system, including in the brain and the central nervous system and the peripheral nervous system. This damage causes progressive deterioration of intellectual functions and motor skills, such as the ability to walk. Affected individuals also develop loss of sensation in the extremities (peripheral neuropathy), incontinence,

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seizures, paralysis, an inability to speak, blindness, and hearing loss. Eventually they lose awareness of their surroundings and become unresponsive. Sulfatide accumulation may also affect other organs and tissues. The most common form of metachromatic leukodystrophy, affecting about 50 to 60% of all individuals with this disorder, is called the late infantile form. This form of the disorder usually appears in the second year of life. Affected children lose any speech they have developed, become weak, and develop problems with walking (gait disturbance). As the disorder worsens, muscle tone generally first decreases, and then increases to the point of rigidity. Individuals with the late infantile form of metachromatic leukodystrophy typically do not survive past childhood. Metachromatic leukodystrophy is reported to occur in 1 in 40,000 to 160,000 individuals worldwide. The condition is more common in certain genetically isolated populations: 1 in 75 in a small group of Jews who immigrated to Israel from southern Arabia (Habbanites), 1 in 2,500 in the western portion of the Navajo Nation, and 1 in 8,000 among Arab groups in Israel. Carrier frequency is approximately 1 in 50 in Yemini Jews and 1 in 100 to 1 in 200 in Israeli Arabs and western part of Navajo Nation in the United States (59-60). Maple Syrup Urine Disease (MSUD) is a rapidly fatal neurodegenerative disease. MSUD is an inborn error of metabolism, resulting from the defective activity of branched-chain α-ketoacid dehydrogenase. The BCKDHB gene is located on chromosome 6q14. The enzymatic defect, transmitted in an autosomal recessive manner, results in an inability to catabolize leucine, isoleucine and valine. MSUD has been described in all ethnic groups and has an estimated worldwide frequency of 1:185,000, however ~30% of families with MSUD are of Ashkenazi Jewish descent. The R183P mutation accounts for most cases of MSUD in Ashkenazi Jews with a carrier frequency of 1:113 (61). Severe Methylenetetrahydrofolate Reductase (MTHFR) Deficiency is an autosomal recessive metabolic disorder of folate metabolism causing elevated plasma homocysteine levels and homocystinuria. The clinical spectrum of severe MTHFR deficiency ranges from the neonatal onset of significant neurological problems to milder adult onset cases. The majority of patients present in the first few years of life with developmental delay and other neurological problems, such as seizures. A carrier frequency of 1:39 was determined in Bukharian Jews for a mutation generating an abnormal splicing and early termination codon (62-64). Nemaline Myopathy (NM) is a slowly progressive or now-progressive neuromuscular disorder characterized by muscle weakness and the presence of rod-shaped structures (nemaline bodies/rods) in affected muscle fibers. The estimated incidence is 1:50,000 live births. NM is found to be caused by mutations in three different genes, of which the most common in Ashkenazi Jewish population is the Nebulin gene mapped to chromosome 2q21.1-2q22. The carrier frequency of this deletion in Ashkenazi Jewish population is 1:108 (65-66). Niemann-Pick Disease type A and B (NPD) is an inborn error of sphingomyelin catabolism that results from the deficient activity of the lysosomal hydrolase, acid. Type A NPD is a severe neurodegenerative disorder of infancy which leads to death by three years of age, whereas Type B NPD has a later age at onset and most patients survive into adulthood. NPD is caused by mutations in the NPC1, NPC2, or SMPD1 gene. The most common mutations in Jewish Ashkenazi population are located to the SMPD1 gene mapped to the chromosomal region11p15.1 to 15.4. Carrier frequency of NPD in Ashkenazi Jewish population is 1:90 to 1:100. Disease frequency is between 1:20,000 and 1:30,000 (67). Progressive Cerebello-Cerebral Atrophy (PCCA) is a newly described autosomal-recessive disease characterized by nondysmorphic profound mental retardation, progressive microcephaly, and severe spasticity. Myoclonic or generalized tonic-clonic seizures are often observed as well. PCCA phenotype was identified in non-consanguineous Jewish Sephardic families particularly in individuals of Moroccan and Iraqi ancestry. Parents of affected individuals are either both of Moroccan ancestry, both of Iraqi ancestry, or of mixed Iraqi-Moroccan ancestry. “Moroccan” mutation differs from “Iraqi” mutation yet carrier frequency is similar and is estimated at 1 in 42 to 43 individuals (68-69).

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Retinitis Pigmentosa (RP-26), also known as rod-cone dystrophies (RCDs), is a group of clinically and genetically heterogeneous retinal disorders, dystrophies, with a worldwide prevalence of 1 in 4000. RP is clinically characterized by retinal pigment deposits (bone spicule–like pigment deposits), nyctalopia ("night blindness"), followed by progressive degeneration of the photoreceptors, visual impairment, which eventually leads to blindness. Mutations in more than 60 genes are known to cause nonsyndromic retinitis pigmentosa. RP26 is caused by homozygous or compound heterozygous mutation in the CERKL gene, which encodes a ceramide kinase, on chromosome 2q31. RP26 is characterized by equally affected cone and rod systems and pronounced macular atrophy. The disorder is common among Yemeni Jews where it causes severe retinal degeneration affecting both rods and cons photoreceptor cells leading to loss of peripheral and central vision.. The carrier frequency among Yemeni Jews is 1 in 22 (70-73).

TMC1 related Nonsyndromic Deafness is caused by mutation in the TMC1 gene on chromosome 9q13-q21. The TMC1 gene provides instructions for making a protein called transmembrane channel-like 1. This protein is found in the inner ear, but its function is not fully understood. Based on its location in the inner ear, the TMC1 protein probably plays a role in converting sound waves to nerve impulses, a critical process for normal hearing. Alternatively, the TMC1 protein may be involved in signaling processes that are important for the survival of cells in the inner ear. Carrier frequency among Moroccan Jews is high, estimated at 1 in 50 individuals with close to 40% of all nonsyndromic deafness occurrences within this ethnic group attributed to mutations in this gene (74-75). Tay Sachs Disease (TSD) is a genetic neurodegenerative lysosomal storage disorder. TSD is fatal in its most common variant known as Infantile Tay-Sachs disease. TSD is inherited in an autosomal recessive pattern. The disease occurs when fatty acid derivative called ganglioside accumulate in the nerve cells of the brain due to deficiency in the activity of the Hexozaminidase A (Hex A) enzyme (77). TSD is caused by mutations on the HEXA gene on chromosome 15. The carrier frequency of TSD is 1:29 in Ashkenazi Jews 1:110 in Moroccan Jews and 1:280 in the general Jewish Israeli population (76). Tyrosinemia Hereditary tyrosinemia type I, also known as Hepatorenal tyrosinemia, Fumarylacetoacetase deficiency and FAH deficiency, is an autosomal recessive disorder caused by deficiency of fumarylacetoacetase (FAH), the last enzyme of tyrosine degradation. The disorder is characterized by progressive liver disease and a secondary renal tubular dysfunction leading to hypophosphatemic rickets. Onset varies from infancy to adolescence. In the most acute form patients present with severe liver failure within weeks after birth, whereas rickets may be the major symptom in chronic tyrosinemia. Left untreated, patients may die from cirrhosis or hepatocellular carcinoma at a young age. The defect in FAH results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. Tyrosinemia type I affects approximately one in 100,000 to 120,000 births. Because of the inconsistent and confusing nature of its clinical presentation, it is estimated that fewer than 50% of affected individuals are diagnosed while alive. In the general US population, the carrier frequency is estimated at 1:150 to 1:100 and among the Ashkenazi Jewish population it is estimated at 1 in 90 (78-79). Usher Syndrome Type 1 constitutes a group of autosomal recessive disorders characterized by progressive pigmentary retinopathy and sensorineural hearing loss. Persons with forms of Usher syndrome type I have congenital severe to profound hearing loss and suffer from Retinitis pigmentosa, a progressive degeneration of the retina, generally appearing in adolescence and leading to night blindness and loss of peripheral vision. The disorders differ in severity with phenotypic distinctions based on auditory and vestibular differences with type1 considered to be the most severe type. The carrier frequency of the defective gene (PCDH15) is 1:100 and the disease frequency is 1:80,000 among Ashkenazi Jewish population (80). Usher Syndrome Type 2A is a clinically and genetically heterogeneous autosomal recessive disorder characterized by sensorineural hearing deficiencies at birth and later development of progressive retinitis

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pigmentosa (RP). It is the most frequent cause of combined deafness and blindness in adults and affects 3 to 6% of children born with hearing impairment. Type II is the most common of the 3 Usher syndromes. Usher syndrome type IIA is caused by homozygous or compound heterozygous mutation in the gene encoding usherin (USH2A) on chromosome 1q41.Patients with Usher syndrome type IIA show moderate to severe sensorineural hearing loss as well as progressive retinitis pigmentosa. Carriage has been identified in Jewish families of Iranian origin. The carrier frequency in this population is estimated in 1 in 26 individuals (81-82). Usher Syndrome Type 3A Unlike the other forms of Usher syndrome, infants with Usher syndrome type III are usually born with normal hearing. Hearing loss typically begins during late childhood or adolescence, after the development of speech, and progresses over time. By middle age, most affected individuals are profoundly deaf. Vision loss caused by retinitis pigmentosa (RP) also develops in late childhood or adolescence, often leading to blindness by mid-life. Individuals with Usher syndrome type III may also experience difficulties with balance due to inner ear problems. These problems vary among affected individuals. The carrier frequency is 1: 107 and the disease frequency is 1: 45,000 among the Ashkenazi Jewish population (83-84). Table 1: Overview of the mutations, diseases and gene name detected by the All In One Kit.

No. Mutation Name Of Disease Full Disease and Gene Name

1 IVS-1 G>C 3MGA (Kostaf) Optic Atrophy Syndrome 3 (OPA3)

2 PiS AAT Alpha 1 Antitrypsin (SERPINA1)

3 PiZ AAT Alpha 1 Antitrypsin (SERPINA1)

4 R35X (c.103C>T) ATM Ataxia Telangiectasia (ATM)

5 2407-2408dupT BLM Bloom Syndrome (BLM)

6 6bp del/7, bp Ins BLM Bloom Syndrome (BLM)

7 693 C>A CAN Canavan Disease (ASPA)

8 854 A>C CAN Canavan Disease (ASPA)

9 1717-1 G>A CF Cystic Fibrosis (CFTR)

10 2751+1insT CF Cystic Fibrosis (CFTR)

11 3121-1 G>A CF Cystic Fibrosis (CFTR)

12 3849+10KB C>T CF Cystic Fibrosis (CFTR)

13 405+1 G>A CF Cystic Fibrosis (CFTR)

14 D1152H CF Cystic Fibrosis (CFTR)

15 F508del CF Cystic Fibrosis (CFTR)

16 G542X CF Cystic Fibrosis (CFTR)

17 G85E CF Cystic Fibrosis (CFTR)

18 I1234V CF Cystic Fibrosis (CFTR)

19 N1303K CF Cystic Fibrosis (CFTR)

20 S549R CF Cystic Fibrosis (CFTR)

21 W1089X CF Cystic Fibrosis (CFTR)

22 W1282X CF Cystic Fibrosis (CFTR)

23 Y1092X CF Cystic Fibrosis (CFTR)

24 Q359K/360K CF Cystic Fibrosis (CFTR)

25 Del2,3 (21Kb) CF Cystic Fibrosis (CFTR)

26 167delIT CNX Non Syndromic Hereditary Hearing Loss (GJB2)

27 35delG CNX Non Syndromic Hereditary Hearing Loss (GJB2)

28 Cx30 CNX Non Syndromic Hereditary Hearing Loss (GJB6)

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29 L90P (c.269T>C) CNXIR Non Syndromic Hereditary Hearing Loss (GJB2)

30 51del12insA CNXUZ Non Syndromic Hereditary Hearing Loss (GJB2)

31 1253delT CTX Cerebrotendinous Xanthomatosis (CYP27A1)

32 IVS4-1 G>A CTX Cerebrotendinous Xanthomatosis (CYP27A1)

33 T339M (c.1016C>T) CTX Cerebrotendinous Xanthomatosis (CYP27A1)

34 G229C DLD Dihydrolipoyl Dehydrogenase Deficiency (DLD or LAD)

35 Y35X DLD Dihydrolipoyl Dehydrogenase Deficiency (DLD or LAD)

36 2173/3insG FANCA Fanconi Anemia Type A (FANCA)

37 4275delT FANCA Fanconi Anemia Type A (FANCA)

38 IVS4+4 A>T FANCC Fanconi Anemia Type C (FANCC)

39 2507+6 T>C FD Familial Dysautonomia (IKBKAP)

40 R696P G>C FD Familial Dysautonomia (IKBKAP)

41 5.5 kb complex deletion GALT Galactosemia (GALT)

42 K285N GALT Galactosemia (GALT)

43 84GG Gaucher Gaucher Type 1 (GBA)

44 IVS2+1 Gaucher Gaucher Type 1 (GBA)

45 L444P Gaucher Gaucher Type 1 (GBA)

46 N370S Gaucher Gaucher Type 1 (GBA)

47 R496H Gaucher Gaucher Type 1 (GBA)

48 RecTL Gaucher Gaucher Type 1 (GBA)

49 V394L Gaucher Gaucher Type 1 (GBA)

50 Q347X (c.1118C>T or c.1039C>T) GSD1a Glycogen Storage Disease Type 1a (G6PC)

51 R83C GSD1a Glycogen Storage Disease Type 1a (G6PC)

52 4455delT GSD3 Glycogen Storage Disease Type 3 (AGL)

53 M712T (c.2186T>C) HIBM Hereditary Inclusion Body Myopathy (GNE)

54 L371P ICCA Infantile Cerebral Cerebellar Atrophy (MED 17)

55 c.35 G>T (R12L) JS Joubert Syndrome 2 (TMEM216)

56 1624delG LGMD2b Limb-Girdle Muscular Dystrophy Type 2b (DYSF)

57 Del(EX1-EX7) ML4 Mucolipidosis IV (MCOLN1)

58 IVS3-2 A>G ML4 Mucolipidosis IV (MCOLN1)

59 G59E (c.176G>A); MLC1 Megalencephalic Vacuolating Leukoencephalopathy (MLC1)

60 P377L (c.2119C>T) MLD Metachromatic Leukodystrophy (ARSA)

61 E372X EXON 10 MSUD Maple Syrup Urine Disease (BCKDHB)

62 G278S EXON 7 MSUD Maple Syrup Urine Disease (BCKDHB)

63 R183P MSUD Maple Syrup Urine Disease (BCKDHB)

64 474 A>T MTHFR Severe Methylenetetrahydrofolate Reductase Deficiency (MTHFR)

65 R2478-D2512del NM Nemaline myopathy (NEB)

66 fsP330 NP Niemann-Pick Disease A (SMPD1)

67 L302P NP Niemann-Pick Disease A (SMPD1)

68 R496L NP Niemann-Pick Disease A (SMPD1)

69 R608 NP Niemann-Pick Disease B (SMPD1)

70 A239T (c.715G>A); PCCA Progressive Cerebello-Cerebral Atrophy (SEPSECS)

71 p.Y334C (c.1001A>G) PCCA Progressive Cerebello-Cerebral Atrophy (SEPSECS)

72 c.238+1G>A RP26 Retinitis Pigmentosa (CERKL)

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73 R389X (c.1165C>T) TMC1 TMC1 related Nonsyndromic Deafness (TMC1)

74 R604X (c.1810X>T) TMC1 TMC1 related Nonsyndromic Deafness (TMC1)

75 S647P (c.1939T>C) TMC1 TMC1 related Nonsyndromic Deafness (TMC1)

76 W404R (c.1210T>C) TMC1 TMC1 related Nonsyndromic Deafness (TMC1)

77 1278InsTATC TSD Tay Sachs Disease (HEXA)

78 DF 304/305 TSD Tay Sachs Disease (HEXA)

79 G269S TSD Tay Sachs Disease (HEXA)

80 IVS12+1 G>C TSD Tay Sachs Disease (HEXA)

81 IVS5-2 A>G TSD Tay Sachs Disease (HEXA)

82 R170Q TSD Tay Sachs Disease (HEXA)

83 R247W (c.C739T) TSD Tay Sachs Disease (HEXA)

84 G250V (c.G749T) TSDIR Tay Sachs Disease (HEXA)

85 L451V (c.C1351G) TSDIR Tay Sachs Disease (HEXA)

86 R393X (c.1177C>T) TSDIR Tay Sachs Disease (HEXA)

87 IVS9+1G>A TSDNJ Tay Sachs Disease (HEXA)

88 P261L TYR Thyrosinemia (FAH)

89 R245X USH1 Usher Syndrome I (PCDH15)

90 c.236_239dupGTAC USH2A Usher Syndrome 2A (USH2A)

91 N48K (c.144T>G) USH3A Usher Syndrome type 3A (CLRN1)

Kit Contents

The All In One Kit contains enough amplification buffer and primer mix for 21 samples and enough detection reagents for two detections run. One to 21 samples can be analyzed in a single detection run. Refer to product package insert for performance characteristics and additional storage information.

Storage

Using NanoChip Cartridges

The All In One Kit is designed to analyze 21 samples on a NanoChip 400 Cartridge. A maximum of six All In

One protocols may be run on a NanoChip 400 Cartridge.

NanoChip Cartridge Handling Handle the cartridge by the outer black housing only; do not touch the clear plastic or electrical contact

area. Exposure to static electricity may damage the cartridge and may affect results. Ensure that the

flowcell window (clear plastic on the underside of the cartridge) is clear of any debris. If debris is present,

REF 800014 800014R

≤-20°C ≤-20°C

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always use a new (not previously opened) Bausch & Lomb Pre-Moistened Tissue to clean the window. DO

NOT use excessive force when wiping the flowcell window. ONLY clean the flowcell window if debris is

present.

Materials and Equipment

Materials Available from Savyon

REF Description Contents

800014 All In One Kit 21 Samples

All In One Amplification Reagents 1 x vial (225 µL) Primer Mix1 1 x vial (225 µL) Primer Mix2 1 x vial (225 µL) Primer Mix3 1 x vial (225 µL) Primer Mix4 2 x vial (850 µL) LS Amplification Buffer

1 x vial (120 µL) NanoChip Taq

All In One Reagent Packs 1 x Reference Reagent Pack1 1 x Reference Reagent Pack2 2 x Capture Reagent Packs1 2 x Capture Reagent Packs 2 2 x Reporter Reagent Packs1 2 x Reporter Reagent Packs2

2 x CAPdown Sample Buffer B

800014R All In One Extra Reagents

All In One Reagent Packs 2 x Capture Reagent Packs1 2 x Capture Reagent Packs2 2 x Reporter Reagent Packs1 2 x Reporter Reagent Packs2

Additional Materials Available from Savyon

REF Description Contents

800160 NanoChip 400 Cartridge 1 cartridge

800161 NanoChip 400 Fluidics Cartridges 4 x fluidics cartridges

800154 NC400 Low Salt Buffer 6 x bottles (25 mL each)

800155 NC400 High Salt Buffer 6 x bottles (25 mL each)

800156 NC400 Target Prep Buffer 6 x bottles (25 mL each)

800061 NanoChip Microplate Seals 100 x 96-well plate seals

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Other Required Materials (not available from Savyon)

Extraction Reagents:

Reagents to extract genomic DNA from blood at a yield > 50 ng/μL

Reagents to run NanoChip® 400 system: L-histidine (Sigma H-8000) Triton® X-100 (Sigma X-100) Water, deionized

Sample Plates

96-well ABI PCR plates (ABI N801-0560)

96-well Thermo-Fast PCR plates (AB-1100)

MicroAmp™ Compression Pads (ABI 4312639)

2µm filters (Nalgene 5660020)

Required Equipment

NanoChip 400 System

Thermal Cycler1

Technical Assistance

Specialists from the Technical Assistance Center can help troubleshoot and resolve problems. Contact the Center via one of the following methods:

E-mail: [email protected]

Phone: +972.8.8562920

Fax: +972.8.8523176

Address: Savyon Diagnostics Ltd.

3 Habosem St. Ashdod 77610 ISRAEL

1 The following models are recommended:

GeneAmp® Thermal Cycler 2700, 2720, or 9700 MJ Research Peltier Thermal Cycler PTC200

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Precautions

Amplification technologies can amplify target nucleic acid sequences over a billion-fold and

provide a means of detecting very low concentrations of target. Care must be taken to

avoid contamination of samples with target molecules from other samples, or amplicons

from previous amplifications. Follow these recommendations to help control

contamination.

1. If possible, isolate pre-amplification steps from post-amplification steps. For example, use separate

rooms for pre- and post-amplification. Each room should contain equipment, such as pipettes,

dedicated to the specific process. Gloves and lab coats should be dedicated to each room as well.

If dedicated rooms are not available, the laboratory should be set up to allow a unidirectional flow.

Prepare samples in a laminar flow hood using dedicated equipment to minimize contamination. Set

up the post-amplification area in a low-traffic area with dedicated equipment.

2. Use disposable containers, disposable barrier pipette tips, disposable bench pads, and disposable

gloves. Avoid washable lab wear.

3. Use a diluted bleach solution (0.2% sodium hypochlorite) to treat waste from the post-amplification

and detection areas, as the waste contains amplicon. Use the bleach solution to wipe down

equipment and bench areas, and to treat drains used to dispose of liquid waste.

4. Monitor contamination with regular swabbing. Use a wet cotton swab to wipe areas of the bench or

equipment, and rinse the swab with 500 µL of water. Test a few microliters of the rinse solution in

the amplification assay to detect possible contamination. If contamination is detected, follow internal

de-contamination procedures.

5. Use negative controls to monitor for possible contamination during reaction setup. If reagent

contamination is detected, dispose of the suspect reagents.

References for Contamination Control

Kwok, S. and Higuchi, R. (1989). Avoiding false positives with PCR. Nature (London) 339, 237.

Victor, T. et al. (1993). Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results. Eur. J. Clin. Chem. Clin. Biochem. 31, 531.

Yap, E.P.H. et al. (1994). False-positives and contamination in PCR. In: PCR Technology: Current Innovations. Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL.

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Performing Sample Amplification

To optimize workflow, you can begin other activities during sample amplification. For example, you can prepare the system and thaw reagents. During cartridge initialization, you can write the protocol and prepare the sample plate.

Extraction

Process a blood sample using an extraction method that yields ≥ 50 ng/µL of genomic DNA.

Amplification

Perform in an amplicon-free area. Four PCR Master Mixes should be generated: Master Mix 1, 2, 3 and 4. 1. Remove the LS Amplification Buffer and the All In One Primer Mixes 1 to 4 from the ≤ -20°C

freezer. Thaw at room temperature and vortex.

Note: The LS Amplification Buffer and the All In One Primer Mixes 1 to 4 may be frozen two additional times, or stored at 2-8º C for one week.

2. Prepare the PCR Master Mixes using the following guidelines per sample

(see Table 2). To ensure an adequate volume of Master Mix, take the number of reactions and add 2. Multiply the sum by the volume of each component shown in Table 1.

Note: Remove the Apta Taq DNA Polymerase from the

freezer immediately prior to use, and return to the freezer promptly after use.

Table 2: PCR

1 Master Mixes Guidelines

Master Mix 1

Component Volume (µL)

LS Amplification Buffer 14.4

All In One Primer Mix 1 7.5

AIO NanoChip TAQ 1.1

Total Master Mix Volume per Reaction 23

1 Refer to Appendix C: Legal Notices, for PCR information.

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Master Mix 2






Master Mix 3






Master Mix 4






3. Add 23 µL of the PCR Master Mix 1 to reaction wells in the PCR plate (from

well A1 to B12). For example, for 8 samples add 23 µL of the PCR Master Mix 1 to reaction wells A1-A8 (See Figure 1).

4. Add 23 µL of the PCR Master Mix 2 to reaction wells in the PCR plate (from well C1 to D12). For example, for 8 samples add 23 µL of the PCR Master Mix 2 to reaction wells C1-C8 (See Figure 1).


well E1 to F12). For example, for 8 samples add 23 µL of the PCR Master Mix 3 to reaction wells E1-E8 (See Figure 1).


well G1 to H12). For example, for 8 samples add 23 µL of the PCR Master Mix 4 to reaction wells G1-G8 (See Figure 1).

7. Add 2 µL of template DNA to the reaction wells of each of the master mixes. For example, add DNA sample 1 to wells A1, C1, E1 and G1 (Figure 1).

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Notes: Do not scale up an amplification reaction; always use 25 µL reaction volumes.

Template DNA must be at least 50 ng/µL.

8. Seal the PCR plate and place into a thermal cycler.

Note: For onboard dilution, cover the 96-well ABI PCR plate with a Nanochip Microplate seal (PN: 800061) and place the plate into the thermal cycler

1.

Place the ABI MicroAmp Compression Pad over the sealed PCR 96-well plate and close the lid of the thermal cycler. Alternatively, the 96-well ABI PCR

plate may be sealed with

standard PCR caps. The caps must be removed and replaced with a Nanochip Microplate seal (PN: 800061) prior to use on the NanoChip 400.

Figure 1: Master mixes and DNA samples location on the 96-well ABI PCR

plate.

9. Program the thermal cycler using the parameters described in Table 3.

1 The following models are recommended:

GeneAmp® Thermal Cycler 2700, 2720, or 9700 MJ Research Peltier Thermal Cycler PTC200

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Table 3: Thermal Cycler Parameters

Temperature (°C) Time Number of Cycles

95 2 minutes 1

95 30 seconds

40 65 1 minute

72 7 minutes 1

4 Hold

10. Once cycling is complete, remove the PCR plate from the thermal cycler. The

prepared plate may be stored at 2-8°C for up to one week, or at ≤ -20°C for up to six months.

Preparing the Sample Plate

Notes: To optimize workflow, begin system preparation, reagent thawing, and creating the

protocol during sample amplification. If not using onboard dilution, prepare the sample plate during cartridge initialization.

The sample dilution option must be set in the All In One template in the Protocol

Editor such that the PERFORM ONBOARD DILUTION setting is checked for automated onboard dilution and unchecked for manual dilution. The template default has the PERFORM ONBOARD DILUTION setting checked for automated onboard dilution.

Option 1 Manual Sample Dilution

1. Remove CAPdown Sample Buffer B from the freezer. Upon thawing, vortex the solution thoroughly

until all precipitates are dissolved.

Note: Once thawed, CAPdown Sample Buffer B can be stored at room temperature or at 2-8°C for up to two weeks. Do not refreeze.

2. For each individual amplification reaction, pipette 62 µL of CAPdown Sample Buffer B into one well of

a 96-well plate. 3. Add 8 µL of each amplification reaction into a well containing CAPdown Sample Buffer B. Carefully

pipette up and down to mix. 4. Cover plate with a Microplate Seal.

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Option 2 Onboard Sample Dilution

1. Remove the ABI MicroAmp™ Compression Pad from the ABI PCR plate covered with Microplate

Seal, attach the plate to the PCR Plate Base and insert into plate position 2 of the NanoChip 400. Or

1. Remove the caps of the ABI PCR plate and replace with a Microplate Seal, attach the plate to the PCR Plate Base and insert into plate position 2 of the NanoChip 400.

Or

1. Pipette a minimum of 20 μL of amplified sample into wells of a 96 well plate 2. Cover with a Microplate Seal and insert plate into plate position 2 of the NanoChip 400.

Note: The Onboard Dilution Option can only be used with the ABI 96 well plate (ABI

N801-0560) attached to the PCR Base Plate. Use of other plate types may cause damage to the instrument.

Operating the NanoChip 400 System

Refer to the NanoChip 400 User’s Guide (REF 140530) for detailed instructions on the basic operation of the system, including system maintenance and cartridge handling.

Preparing Solutions for Use in the NanoChip 400 Instrument

The following table describes the required solutions, and their assigned locations within the instrument.

Table 4: Location of Bottles in the NanoChip 400 Instrument

Solution Bottle Location Minimum Volume*

Water 1 L H2O position 400 mL

Wash Solution 1 L BUF position 400 mL

High Salt Buffer 30 mL Position 1 25 mL

Low Salt Buffer 30 mL Position 2 25 mL

Target Prep Buffer 30 mL Position 3 25 mL

**CAPdown Sample Buffer B

30 mL Position 4 25 mL

* The minimum volume of liquid that should be in the listed bottle before starting the

assay run

**CAPdown Sample Buffer B is only required if performing onboard dilution.

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Preparing the Wash Solution Preparing the Wash Solution for NC400 Instrument.

1. 50 mM histidine solution

In a bottle/beaker, add 7.76 g of L-histidine to a final volume of 1 L of dH2O for 50 mM histidine. Mix until histidine is dissolved. Filter

the solution using a 0.2 m filter.

Note: This solution is stable for up to two week at 2–8oC.

2. 20% Triton X-100 solution

a. Add 4 mL or 4.24 g of Triton X-100 to approximately 16 mL of dH2O for a final volume of 20 mL.

b. Mix solution thoroughly (approximately 10 minutes).

Note: This solution is stable for up to three months at 2-8oC.

3. Combine component solutions daily to make fresh wash solution

(50 mM histidine, 0.1% Triton X-100). a. Add 400 mL of the 50 mM histidine solution to a 1 L buffer

bottle. b. Add 2 mL of the 20% Triton X-100 solution and mix

thoroughly. Note: Make wash solution fresh daily.

Preparing the NanoChip Cartridge and Instrument

1. Remove the following reagent pack from the freezer and place at room temperature

to thaw.

All In One Capture Reagent Packs 1 and 2 All In One Reporter Reagent Packs 1 and 2

All In One Reference Reagent Pack 1 and 2 Notes: The reagent pack must be used within 8 hours of thawing.

Because the item listed above is single use only, discard after use.

The All In One Reference Reagent Pack is only required for the first use of a cartridge.

2. Remove a NanoChip Cartridge from 2-8°C storage. Keep at room temperature for

at least 15 minutes before using. Note: Bringing the cartridge to room temperature before insertion into the

instrument avoids the formation of condensation in the cartridge window, which could cause the cartridge to fail initialization.

3. Initialize and prime the NanoChip 400 Instrument following the guidelines listed in the NanoChip 400 User’s Guide.

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4. From the Dock Bar, select the instrument icon to start the NanoChip 400 Instrument

Manager.

5. Ensure that the flowcell window (clear plastic on the underside of the cartridge) is clear of any debris. If debris is present, use a new (not previously opened) Bausch & Lomb Pre-Moistened Tissue to clean the window.

Note: Do NOT use excessive force when wiping the flowcell window. Clean the

flowcell ONLY when debris is present.

6. Scan the barcode of the NanoChip Cartridge using the attached barcode scanner.

Note: The barcode will not display in the Instrument Manager until step 8 has been completed.

7. Insert the cartridge into the instrument, ensuring that it is properly seated.

8. Close the cartridge door by pressing the button located below the cartridge slot on the instrument.

9. When the Cartridge Initialization window appears, select Initialize Cartridge with Hydration.

10. Cartridge initialization will take approximately 15 minutes. When initialization is

completed, the LCD will display “Instrument Ready”.

11. Write the protocol as described in the following section.

Note: The protocol can be written while the cartridge is initializing.

Creating a Protocol

Using the Protocol Editor, create the following protocol to address and report 1-96 samples.

Create a new protocol for each sample run. For detailed instructions on using Protocol

Editor, see the NanoChip 400 User’s Guide.

1. From the Dock Bar select Protocol Editor.

2. Select Create A New Protocol; select OK.

3. Select the All In One icon from the available templates

Note: The All In One template automatically determines prior pad utilization, and maps capture and sample addressing beginning with the first unused sample position.

4. The Plate Specification Window appears; choose the correct plate type intended for the assay from the options in the pull-down menu. Select OK.

Note: Selecting a sample plate type other than what is placed on the NanoChip 400 Instrument deck at the start of a run can cause damage to the system and fail the run. Use caution to select the appropriate plate type.

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5. The Set Cartridge window appears; choose Select The Cartridge. From the pull-down menu, select the serial number of the cartridge that will be used in the run (or type the serial number into the window). Select OK.

Note: If the cartridge selected is still initializing, a cartridge presently in use window will appear. Select Yes to indicate that you still want to use this cartridge for the protocol you are creating.

Warning: Select No if the cartridge selected is in use with a All In One Protocol and wait for the protocol to complete before creating a new All In One protocol for the selected cartridge. If Yes is selected, the pad usage for the new protocol may not map correctly.

Note: A maximum of six All In One protocols may be run on a NanoChip 400 Cartridge. After all test sites on the cartridge have been used once with the All In One protocol, the cartridge may be reused with the All In One protocol a maximum of three times.

Figure 2. All In One Temporary Protocol

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6. Select Materials Configuration and enter sample names manually, or select Import Content to import sample information from a Plate Content Definition Microsoft® Office Excel template. You may also enter information into the Description box if desired.

Note: Be sure sample names entered correspond to the wells used in setting up the sample plate.

If the number of samples exceeds the available sample positions on the

cartridge, the software will notify the user.

7. Select the All In One step in the Protocol Structure tree. Select only the well containing primer mix 1 that will be used (Bold) (see Fig. 2). Wells that contain primer mix 2, 3 and 4 will be automatically updated during the file conversion (see Fig. 3). Marked wells in the 96-well plate display will have a dot. Note that the number of wells equals the number of DNA samples to be analyzed.

Notes: The sample dilution option must be set in the All In One template in

the Protocol Editor such that the PERFORM ONBOARD DILUTION setting is

checked for automated onboard dilution and unchecked for manual

dilution. Multiple wells may be selected simultaneously by clicking the row (A – H) or

the column (1 – 12) on the sample plate in the All In One template.

8. Select File/Save As from the command tool bar. Enter a name for the protocol and save it.

9. Double click the file convertors icon on your desktop.

- Click the "Select" icon and choose the file saved in section 8.

- Click the "Convert" icon , the convertor will automatically convert the file.

- Click the "Save" icon and save the final format under a different name.

- Click the "exit" icon . Your file is now ready to be loaded.

Notes: Any change to the protocol must be done on the file saved in section 8 and conversion (Stage 9) must be done again.

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Figure 3. All In One Final Protocol

Running the Assay

1. From the Instrument Manager, select Open from the Manager Panel screen. Browse to

select the protocol file you just created.

2. Note: When running a re-use protocol a dialog box will appear warning the user that pads have been previously addressed and requires a password to continue. Scroll to the bottom of the dialog to obtain the necessary password

3. The system calculates the amount of waste the protocol will generate. Check the waste bottle, making sure it has enough room to hold the new waste. If there is room, select Continue. If the waste bottle does not have enough room, empty it before running the assay, and then select Continue.

4. Load reagents on the instrument deck

a. Place the following buffer bottles on the instrument deck as instructed by the Instrument LCD prompts.

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Table 5: Location of Bottles in the NanoChip 400 Instrument

Solution Bottle

Size

Location

High Salt Buffer 30 mL Slot 1

Low Salt Buffer 30 mL Slot 2

Target Prep Buffer 30 mL Slot 3

CAPdown Sample Buffer B* 30 mL Slot 4

*Required for Onboard Sample Dilution option only. This position is left

empty when sample dilution is done manually.

b. Place the Reagent Pack Plate in Plate Location 1 of the instrument deck as instructed by

the LCD prompt.

Reagent packs are loaded into a Reagent Pack Plate before they are placed in the instrument deck as follows: All In One Capture Reagent Pack 1 (FTA) – Position 1 All In One Capture Reagent Pack 2 (FTB) – Position 2 All In One Reporter Reagent Pack 1 (FTC) – Position 3 All In One Reporter Reagent Pack 2 (FTD) – Position 4 All In One Reference Reagent Pack 1 (FTE) – Position 5 All In One Reference Reagent Pack 2 (FTF) – Position 6

c. Place the sample plate in Plate Location 2 of the instrument deck as instructed by LCD prompt.

Notes: When using an ABI 96-well sample plate on deck, always position the plate with

well A1 in the upper left-hand corner. 5. After the run is complete, select Eject from the Instrument Manager screen. When the

LCD displays “Remove Cartridge”, remove the cartridge from the instrument. If the cartridge has not been fully used, return the cartridge to its pouch and store at 2-8°C. If the cartridge has been fully used, discard it. Note: When the eject button is selected, a window will appear asking the user to strip and/or fill

the cartridge before ejecting. Select Fill scroll down and choose Water. 6. Remove all buffers and replace the Wash Buffer with water. Perform routine

maintenance as appropriate.

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Analyzing the Data

A detailed description of the assay format can be found in Appendix A. Briefly, the genotyping calls are based on a green-to-red ratio where green indicates the presence of the wild type allele, and red the presence of the mutant allele. The data are analyzed in a Microsoft Office Excel based spreadsheet. Refer to Appendix B for a description of the All In One Data Analysis Spreadsheet features, instructions for setting preferences, and data calculations. 1. Export the data from a All In One NanoChip 400 run as follows:

a. Select Data Analysis from the NanoChip 400 Dockbar.

b. Select Export Processed Data. Select Next.

c. Select the appropriate cartridge and session number. The session numbers are listed by date, followed by the time the assay run started.

d. Select all 20 green and all 20 red image data files; select Finish.

e. A new screen displays. In the View tab, select Show Non-Activated Pads.

f. Select Export on the lower right side of the NanoChip 400 Data Analysis window.

g. A new screen appears; be sure all the boxes are checked and select Export.

h. Enter a file name (for example, the cartridge serial number and date of the run) and select Save. An Excel spreadsheet is automatically generated.

i. Close the NanoChip 400 Data Analysis software.

2. Import the All In One data into the All In One Data Analysis Spreadsheet

a. Open the All In One Data Analysis Spreadsheet.

b. Select the Import button. Find the file you just saved and select Open.

c. A new message appears that prompts the user to save the Data Analysis Spreadsheet. A default name of “cartridge number session number” is given, but another name may be assigned.

Notes: If Show Non-Activated Pads was not selected during data export, an error message will appear when data import is attempted to the All In One Data Analysis Spreadsheet. If this occurs, repeat the data export process with the Show Non-Activated Pads selected.

To prevent data overwriting, the Import button is removed after a set of data is imported.

d. Save your changes to the spreadsheet.

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Appendix A: All In One Assay Format

Assay Format

The All In One assay uses a capture down format to genotype the markers based on identified sample. Following the single tube multiplex polymerase chain reaction, the amplicons are specifically bound to a permeation layer that covers the electronic microarray via hybridization to complementary capture oligonucleotides. These capture oligonucleotides are biotinylated at the 5’ or 3’ end and are bound to streptavidin that has been incorporated into the permeation layer. The All In One Kit components include the following:

All In One Primer Mixes 1-4: set of forward and reverse amplification primers that specifically amplify segments of 36 different genes. LS Amplification Buffer: a general-purpose reagent used for the PCR amplification of DNA in an ionic environment optimized for analysis on the NanoChip 400 electronic microarray.

All In One Capture Reagent Packs (1 and 2) contains a set of 18 unique capture mixes. Each capture is a biotinylated synthetic oligonucleotide complementary to one of the amplicons generated with the All In One primer mixes. Each capture is present in one of the eighteen capture mixes. All In One Reporter Reagent Packs (1 and 2) contains 20 unique reporter mixes. Reporter mixes contain discriminators and universal reporters. Each discriminator contains a segment that is complementary to the wild type or mutant allele. Those with a wild type complement also contain a segment that is complementary to the “wild type” universal reporter that contains a green fluorophore. Those with a mutant complement also contain a segment that is complementary to the “mutant” universal reporter that contains a red fluorophore. Each All In One reporter mixes contains numerous pairs of discriminators. All In One Reference Packs (1 and 2) contains a set of 18 unique mixes of biotinylated reference oligonucleotides. The reference oligonucleotides have a segment complementary to one or more discriminator oligonucleotides. The green and red signals generated from the references indicate the reporter mixes and reporting protocol are working properly. CAPdown Sample Buffer B: a general-purpose reagent used for the delivery of amplicons to the activated test sites on the NanoChip 400 electronic microarray.

Starting with the amplified material, the All In One protocols generated as described in the “Creating a Protocol” section consist of the following five distinct steps.

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1. Capture addressing: the capture oligonucleotide mixes specific for the All In One assay are electronically addressed to predetermined pads across the cartridge in a sequential manner. The number of pads addressed with each mix is equal to the number of samples/controls being analyzed. Wells 1–18 of the All In One Captures Reagent Pack contain Capture Mixes 1–18.

2. Reference addressing: the reference oligonucleotide mixes specific for the All In One assay are electronically addressed to predetermined pads in the NanoChip microarray. Each reference mix is addressed in two separate electronic activation events to separate pads. References are addressed in the first use of the cartridge— subsequent cartridge runs utilize references addressed in the first use. The reference mixes are in wells 1–18 of the All In One Reference Reagent Pack

3. Amplicon Hybridization: amplification reaction products diluted in CAPdown Sample Buffer B are

simultaneously addressed to 18 pads that comprise the full set of the Capture Mixes 1-18. The amplicons are sorted across the 18 pads by hybridization to specific captures. An amplicon hybridizes to just 1 of the 18 capture pads.

4. Reporting: sequential cycles of passive hybridization-thermal discrimination-fluorescence

imaging-thermal stripping ensue for each of the 20 reporter mixes contained in the All In One reporter Reagent Pack. The thermal stripping step removes the discriminator/universal reporters but leaves the amplicon bound to the capture oligonucleotide for the next reporter mix. Well 1-20 of the All In One Reporter Reagent Pack contain Reporter mixes 1 to 20.

5. Reverse Bias Washing: each pad that was addressed with sample is subjected to a reverse

biasing to remove bound amplicon that can potentially interfere with future assays on the microarray. After Reverse Bias Washing, the system automatically fills the cartridge with Water for storage between uses.

The following tables and figures map the capture, sample, and reference pad locations to the 16 X 25 array of the NanoChip Cartridge. Numbers 1-20 in Table 6 refer to the sample position of sequentially addressed samples. Note that each sample is addressed to 18 pads.

Table 6 maps the location of the capture mixes within the 18 pads of each sample. The All In One template automatically maps samples starting with the first available sample position: for the first use of a cartridge, the first sample is addressed to sample position 1; if 10 sample positions were used in the first All In One run, the second use will begin with sample position 11. The All In One template automatically maps the capture mixes to the sample positions that are being used in the current All In One run. The sample position in the All In One Data Analysis Spreadsheet is referenced in three locations: the Samples Worksheet, Summary Worksheet, and Data Table Worksheet.

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Table 6 : Capture, Sample and Reference Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

1 1 1 12 2 13 3 14 4 15 5 5 16 6 R9 17 7 18 18 8 19 9 20 10 21 11

2 1 1 12 2 13 3 14 4 15 5 5 16 6 R10 17 7 18 18 8 19 9 20 10 21 11

3 1 12 12 2 13 3 14 4 15 5 16 16 6 R11 17 7 18 8 8 19 9 20 10 21 11

4 1 12 12 2 13 3 14 4 15 5 16 16 6 R12 17 7 18 8 8 19 9 20 10 21 11

5 1 12 2 2 13 3 14 4 15 5 16 6 6 R13 17 7 18 8 19 19 9 20 10 21 11

6 1 12 2 2 13 3 14 4 15 5 16 6 6 R14 17 7 18 8 19 19 9 20 10 21 11

7 1 12 2 13 13 3 14 4 15 5 16 6 R15 17 7 18 8 19 9 9 20 10 21 11

8 1 12 2 13 13 3 14 4 15 5 16 6 R16 17 7 18 8 19 9 9 20 10 21 11

9 1 12 2 13 3 3 14 4 15 5 16 6 R1 R17 17 7 18 8 19 9 20 20 10 21 11

10 1 12 2 13 3 3 14 4 15 5 16 6 R2 R18 17 7 18 8 19 9 20 20 10 21 11

11 1 12 2 13 3 14 14 4 15 5 16 6 R3 17 7 18 8 19 9 20 10 10 21 11

12 1 12 2 13 3 14 14 4 15 5 16 6 R4 17 7 18 8 19 9 20 10 10 21 11

13 1 12 2 13 3 14 4 4 15 5 16 6 R5 17 17 7 18 8 19 9 20 10 21 21 11

14 1 12 2 13 3 14 4 4 15 5 16 6 R6 17 17 7 18 8 19 9 20 10 21 21 11

15 1 12 2 13 3 14 4 15 15 5 16 6 R7 17 7 7 18 8 19 9 20 10 21 11 11

16 1 12 2 13 3 14 4 15 15 5 16 6 R8 17 7 7 18 8 19 9 20 10 21 11 11

Capture 1

Capture 2

Capture 3

Capture 4

Capture 5

Capture 6

Capture 7

Capture 8

Capture 9

Capture 10

Capture 11

Capture 12

Capture 13

Capture 14

Capture 15

Capture 16

Capture 17

Capture 18

R1 = All In One Reference 1, R2 = All In One Reference 2, etc.

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Figure 4 A-C displays the maps of four PCR mixes including markers reported across the 20 reporting mixes. Each reporter mix reports markers across the 18 sample pads. The unused pad serves as the background for that reporting. Each sample has its own background pad. The pad used for the background in each reporting is designated “CONTROL” in the figure. For example, Reporter mix 1 reports F508del from PCR 1 on capture pad 1, 1717-1 G>A from PCR 1 on pad 2, N1303K from PCR 1 on pad 3, 3849+10kb from PCR 1 C>T on pad 4, G250V from PCR 2 on pad 12, A239T from PCR 3 on pad 14, R35X from PCR 3 on pad 16 and G59E from PCR 4 on pad 17. No markers are reported on capture pads 4, 6-11, 13, 15 and 18. Pad 9 is marked CONTROL and used as the background pad in data calculations (see details in Appendix B). 4A. Multiplex PCR 1

Pad 1 Pad 2 Pad 3 Pad 4 Pad 5 Pad 6

Capture Mix 1 Capture Mix 2 Capture Mix 3 Capture Mix 4 Capture Mix 5 Capture Mix 6

Reporter 1 ΔF508 1717-1 G>A N1303K

3849+10kb C>T

Reporter 2 G85E G542X I1234V W1282X

Reporter 3 3121-1G>A S549R (T>G)

Q359K/ T360K W1089X CFTR del2,3

Reporter 4 405+1 G>A D1152H Y1092X

Reporter 5 Control

Reporter 6 IVS2+1 IVS-1 G>C

Reporter 7

Reporter 8 Control

Reporter 9 35delG Control

Reporter 10 84InsG 1624delG

Reporter 11 Control

Reporter 12

Reporter 13 V394L 2172InsG

Reporter 14 N370S

Reporter 15 1253delT P261L

Reporter 16 Control Cx30

Reporter 17 RecTL

Reporter 18 167delT

Reporter 19 474 A>T

Reporter 20

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4B. Multiplex PCR 2



Reporter 1 Control

Reporter 2 Control

Reporter 3 G218T

Reporter 4 NPA-R496L Control

Reporter 5

IVS12+1 G>C R247W

Reporter 6 MSUD-R183P FD-R696P

Reporter 7 Bloom 6 del/7 ins NPA-L302P

CAN-693 C>A

FD-2507+ 6 T>C

Reporter 8 NPB-DR608

Nemaline 24D25P

ML4-IVS3-2A>G

Reporter 9 R393X

USHT1-R245X

FAC-IVS4+4A>T G250V

Reporter 10 Control AAT-PIZ

Reporter 11

CAN-854 A>C

Reporter 12 Control

Reporter 13 IVS5-2 A>G Control

Reporter 14 R170Q G>A Control

Reporter 15 1278insTAT

C ∆F304/305 G269S

Reporter 16 PIS

GSD1A-R83C

Reporter 17 Control

Reporter 18 L451V

Reporter 19 Control

Reporter 20 Control

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4C. Multiplex PCR 3 (pads 13-16) +4 (pads 17-18)



Reporter 1 A239T R35X G59E

Reporter 2 T339M

Reporter 3 Control

Reporter 4 K285N L371P

Reporter 5 51del12InsA

ML4-Del [ex 1-7]

Reporter 6 IVS9+1 Control Control

Reporter 7 Control

NPA-fsP-330

Reporter 8

Reporter 9 4455delT

236_239dupGTAC G229C

Reporter 10

Reporter 11 W404R 238+1 G>A M712T

Reporter 12 IVS4-1 2751+1InsT R496H

Reporter 13 S647P

Reporter 14 R389X DEL 5.5

Reporter 15 Control

Reporter 16 2407dupT Q347X

Reporter 17 4275 delT Y334C P377L

Reporter 18 L90P Y35X N48K

Reporter 19 R604X G278S L444P

Reporter 20 E372X

Figure 4A-C: Distribution maps of the Reporter Mixes 1–20 Across Capture Pads 1–18 and four PCR mixes

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Appendix B: All In One Data Analysis Spreadsheet and Data Calculations

Getting Started The security and preferences for the Data Analysis Spreadsheet require setting the first time the sheet is used. Security Setting The All In One Data Analysis Spreadsheet is a Microsoft Excel Workbook; imported data are calculated to results and genotyping calls using a macro. The Excel security setting must be set to medium or low to allow the use of macros. To adjust the security setting, open Microsoft Excel and select Options from the Tools menu in Excel. Under the Security tab, select the Macros Security box and select Medium. Select Ok. Always select Enable Macros when prompted. Read Only The All In One Data Analysis Spreadsheet is a Read-Only file and will prompt the user to save the file with a new name when preferences are set. Preference Setting

1. Information Header

Open the All In One Data Analysis Spreadsheet. Enter information for the header where prompted on the Samples Worksheet. The information header will appear on every worksheet and on every printed page.

2. Save Settings

Select File/Save As and save your preferences with a new file name.

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All In One Worksheets Samples Worksheet

The sample ID, cartridge number, cartridge session number, operator ID and instrument ID are imported to the Samples Worksheet. The Sample IDs and Sample ethnicities may be edited on this sheet. Boxes for the information header and comments are provided. All other cells are protected and cannot be edited. A footer with lines for “Reviewed By” and “Approved By” is on

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