DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en santé et en services sociaux (INESSS), and that version can be consulted at https://www.inesss.qc.ca/fileadmin/doc/INESSS/Analyse_biomedicale/Juin_2014/INESSS_Avis_ministre_analyses_ biologie_medicale_juin_2014_2.pdf. It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS’s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation. While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation. Four Recessive Diseases of Saguenay–Lac-Saint-Jean — Mutations Screening (Reference 2013.03.011) Notice of Assessment June 2014
Transcript
DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en santé et en services sociaux (INESSS), and that version can be consulted at https://www.inesss.qc.ca/fileadmin/doc/INESSS/Analyse_biomedicale/Juin_2014/INESSS_Avis_ministre_analyses_biologie_medicale_juin_2014_2.pdf. It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS’s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation.
While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation.
Four Recessive Diseases of Saguenay–Lac-Saint-Jean —
Mutations Screening (Reference 2013.03.011)
Notice of Assessment
June 2014
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1 GENERAL INFORMATION
1.1 Requesters: CSSS de Chicoutimi – CHA régional
1.2 Application for Review Submitted to MSSS
September 3 and 27, 2013: qualitative analysis with multiple amplification followed by the detection of mutations by direct allele-specific hybridization of amplicons.
January 29, 2014: new technology proposed: amplification testing followed by the detection of mutations using direct allele-specific hybridization of amplicons (TaqMan® technology).
1.3 Application Received by INESSS: November 1, 2013, and January 30, 2014
1.4 Notice Issued: June 30, 2014
Note:
This notice is based on the scientific and commercial information submitted by the requester and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS.
2 TECHNOLOGY, COMPANY, AND LICENCE(S)
2.1 Name of the Technology
The screening of four recessive diseases in Saguenay–Lac-Saint-Jean: congenital lactic acidosis (CLA-SLSJ), hereditary tyrosinemia type 1 (HT1), hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), and autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). Screening using TaqMan® technology, which can determine the presence or absence of mutant alleles corresponding to one of the four diseases. Five mutations are sought for the four diseases, as two mutations are specific to ARSACS.
2.2 Brief Description of the Technology, and Clinical and Technical Specifications
"The amplification of the genomic regions containing the five target mutations is performed by polymerase chain reaction (PCR). Each mutation requires an amplification reaction. The resulting amplicons are detected using specific TaqMan® probes (Life Technologies/Applied Biosystems) at each of the 10 possible alleles. Once the amplification is completed, the samples are analyzed using an ABI 7500 Fast (Life Technologies) real-time thermal cycler, which measures the fluorescence emitted by each of the 10 probes. Whether or not an amplicon is hybridized on each of the 10 probes determines the presence or absence of fluorescence. This test, based on the Watson-Crick base pairing principle, is highly specific. The principle is widely used in molecular genetics." (requesters' form: details on the genes and technique).
This PCR technique with hydrolysis probes (or TaqMan® assay), used for the identification of genetic polymorphisms, is a versatile and individualized system, as the company can synthesize primers and probes independently and prepare made-to-order assays. It will therefore be easy to rapidly identify the natural alleles of carriers of a mutation using automated fluorescence. A product bulletin by Life Technologies, TaqMan® SNP Genotyping
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Assays [Life Technologies, 2011], describes the technology, and it is also reported in the literature [De Kok et al., 2002]. Appendix A provides a brief description (by Nathalie Girard) and illustration (Life Technologies) of the technology.
2.3 Company or Developer
In-house technique. Local development following a pilot study that began in 2009 and was completed in 2012, in collaboration with the Centre hospitalier universitaire de Québec (CHUQ). The requester provided a standard operating procedure.
2.4 Weighted Value: 28.3 with the old technology and 23.0 with TaqMan® technology.
3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES
3.1 Targeted Patient Group
Tests used to detect mutations associated with four recessive diseases found in Saguenay–Lac-Saint-Jean (SLSJ) targets individuals residing in SLSJ who have at least one grandparent born in Saguenay–Lac-Saint-Jean, Charlevoix or the Haute-Côte-Nord region.
This test was specifically designed for women who are less than 14 weeks pregnant or for couples 18 years of age and older who wish to have children.
3.2 Targeted Disease(s)
Table 1 presents the key aspects and features of the four diseases being diagnosed. The abbreviations used are described in Appendix B.
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Table 1: Key aspects and features of the four diseases
NAME OF DISEASE CONGENITAL LACTIC ACIDOSIS,
SAGUENAY–LAC-SAINT-JEAN TYPE
CONGENITAL, HEREDITARY TYROSINEMIA TYPE I
(SPECIFIC TO SLSJ)
HEREDITARY MOTOR AND SENSORY NEUROPATHY WITH OR
WITHOUT AGENESIS OF THE CORPUS CALLOSUM
AUTOSOMAL RECESSIVE SPASTIC ATAXIA OF
CHARLEVOIX-SAGUENAY
Acronym CLA SLSJ HT1 HMSN/ACC ARSACS
Orphanet number 70472 882 14059 98
Worldwide prevalence Unknown 1/120,000 [Bergeron et al., 2001]
Very rare (2 cases) Unknown
Prevalence in SLSJ 1/2,000 births 1/1,846 [Bergeron et al., 2001] 1/2,117 births 1/1,932 births
Carriers in SLSJ 1/23 1/20 [Bergeron et al., 2001] 1/23 1/22
Chromosome 2p21 15q23-25 15q14 (localized in 1996, identified in 2002)
13q11
Protein LRPPRC (2003, 4, 6) Still unknown sacsin
Type of disease Mitochondrial and metabolic disease
Inborn error of AA metabolism Type of muscular dystrophy caused by a degeneration of the peripheral nerves: impaired body movements, perception and sensation
Neurodegenerative disease characterized by early onset cerebellar ataxia, spasticity and peripheral neuropathy
Genetic diagnosis and other
Ala354Val mutation, particularly lactate in the blood or cerebrospinal fluid (CSF)
Early infantile form (0 to 5 months): hepatocellular necrosis with vomiting, diarrhea, hypoglycemia, edema and renal tubulopathy Late-onset form: vitamin-resistant rachitism, polyneuritis, dystonia, frequent malignant hepatomas
< 1 year: muscle weakness, delayed motor development Later stage: loss of mobility, limb deformities, intellectual disability
12 to 18 months: gait impairment, dysarthria, nystagmus, pyramidal disease, deep sensitivity deficits
Treatments Not specific to the disease (special diet, all day)
Nitisinone (NTBC) Liver transplantation
Incurable (death between 29 and 33 years) Support, comfort
Symptomatic treatment, physiotherapy, wheelchair up to 50-60 years
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3.2.1 Congenital Lactic Acidosis, Saguenay–Lac-Saint-Jean Type
Congenital lactic acidosis specific to Quebec belongs to a group of diseases known as encephalopathy and Leigh syndrome. It is also referred to in literature as Leigh syndrome, French-Canadian type; Leigh syndrome, Saguenay–Lac-Saint-Jean type; and cytochrome-c oxidase deficiency, French-Canadian type [Morin, 2011].
Worldwide prevalence is unknown. In Saguenay–Lac-Saint-Jean, the disease affects 1 out of 2,000 births, whereas 1 out of 23 individuals is a carrier of the genetic mutation (Table 1).
In 2003, the genetic cause of lactic acidosis specific to the region was discovered on chromosome 2: the LRPPRC (leucine-rich pentatricopeptide repeat containing protein) gene.
In 2004, it was confirmed that mutations in the LRPPRC gene decrease the production of cytochrome-c oxidase (COX) and cause the accumulation of lactic acid in the blood. The protein's role in mitochondria was discovered in 2006. In more than 95% of cases, there is a A354V mutation in chromosome 2(2p21). The disease is characterized by chronic metabolic acidosis, hypotonia, facial dysmorphism (prominent forehead, low hairline, hypertelorism, and arched eyebrows, among others), delayed psychomotor development, and moderate intellectual disability. Saguenay-Lac-Saint-Jean (SLSJ) type congenital lactic acidosis is characterized by episodes of metabolic acidosis that can be triggered by infection, intense physical exercise, prolonged fasting, or intense psychological stress. The accumulation of lactic acid decreases blood pH; therefore, to compensate for acidemia, children breathe very deeply and hyperventilate. This may cause coma and pulmonary edema [Ouellette, 2008].
The disease is generally diagnosed between the ages of 0 and 4 years. There are three forms of SLSJ type lactic acidosis, corresponding to varying degrees of severity: 1) the neonatal form is characterized by fulminant acidotic states; 2) the classic form can appear as severe lactic acidosis (from birth) or as ataxic gait (between 14 months and 24 months) ; 3) the "survivor" form occurs in patients who have survived several episodes and show varying degrees of hypotonia, asthenia, developmental delay and, in older patients, truncal ataxia and a characteristic gait. There is no specific treatment for this condition, and patients with the disease are encouraged to eat several small meals throughout the day to reduce the high energy demands of digestion [Morin, 2011].
The definitive diagnosis is established with a genetic test that detects the mutation specific to the disease. The concentration of lactate levels in blood or cerebrospinal fluid may also be measured. Lastly, activity of COX enzymes can be measured in fibroblasts (Table 1).
3.2.2 Hereditary Tyrosinemia Type 1 (HT1), Specific to Saguenay–Lac-Saint-Jean
Hereditary tyrosinemia type 1 is a metabolic disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme in tyrosine catabolism. This enzyme is found mainly in the liver and kidneys, which explains why the pathological effects arise primarily in these two organs [AETMIS, 2007].
Although HT1 has been part of Quebec's Newborn Blood Screening Program since 1970, parents and adults at high risk are not screened.
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According to Bergeron et al. [2001], the worldwide prevalence of hereditary tyrosinemia type 1 is 1 in 120,000 births. However, the prevalence is much higher in Saguenay–Lac-Saint-Jean, where approximately 1 in 1,846 newborns is affected and 1 in 20 individuals is a carrier (Table 1). The very high prevalence in this region is the result of a founder effect involving primarily mutations in intron 12 (IVS12+5G>A-splice mutation), which accounts for approximately 95% of the mutations [Bergeron et al., 2001].
The FAH gene is located in the q23 – q25 region of chromosome 15 (Table 1).
Two clinical forms (acute and chronic) have been reported. The acute form, which is more severe, accounts for the majority of cases and generally appears at two weeks to three months, although it may become apparent during the first hours after birth. The disease progresses rapidly, causing severe liver damage (hepatocellular necrosis) and renal tubular dysfunction. Signs of hepatic failure are vomiting, diarrhea, icterus, hypoglycemia, edema, ascites and coagulation abnormalities [Grompe, 2014; De Lonlay, 2007]. Rapid deterioration is often observed, followed by early death. The chronic form, involving mixed cirrhosis (micronodular and macronodular) and a high risk of hepatocellular carcinoma, affects up to 37% of patients over the age of 2 [Grompe, 2014]. The disease may also have a late onset and manifest as hypophosphatemic rickets or vitamin D-resistant rickets associated with tubulopathy [De Lonlay, 2007]. Very severe neurological manifestations are common when treatment is suboptimal, increasing mortality and morbidity rates. These neurological episodes, resembling those of porphyric neuropathy, are characterized by severe pain with extensor hypertonia, vomiting, paralytic ileus, muscular weakness, and self-mutilation [Grompe, 2014].
The diagnosis is confirmed with genetic testing for known or suspected mutations. A founder mutation is responsible for the high prevalence in the Saguenay–Lac-Saint-Jean region (Table 1). The diagnosis is also confirmed by the detection of aminolevulinic acid in urine, while gas chromatography detects the presence of urinary succinylacetone. Enzymatic assays in fibroblasts may also be included [De Lonlay, 2007].
Previous treatment consisted in following a very strict diet low in tyrosine, phenylalanine and methionine. Current pharmacological treatment consists in administering an inhibitor of hydroxyphenylpyruvate dioxygenase, an enzyme that is upstream of FAH [AETMIS, 2007]. This medication, called nitisinone or NTBC (2-(2-nitro-4- trifluoromethylbenzoyl)-1, 3-cyclohexanedione), has been available since 2005 and has allowed major advances to be made in the treatment of patients with hereditary tyrosinemia type 1 when it is combined with a strict diet and close monitoring of disease progression [De Laet et al., 2013]. Liver transplantation is an option if complications occur or if therapeutic response is not achieved [Grompe, 2014; De Lonlay, 2007].
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3.2.3 Hereditary Motor and Sensory Neuropathy with or without Agenesis of the Corpus Callosum
Hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC) is a progressive neuromuscular disorder that mainly affects people from the Saguenay–Lac-Saint-Jean and Charlevoix regions [Muscular Dystrophy Canada, 2013].
HMSN/ACC causes the rapid degeneration of the peripheral nerves involved in body movement and the perception of sensations (Table 1). Abnormalities are observed in the brain of people with the disease, mainly in the corpus callosum, the structure separating the two cerebral hemispheres. The corpus callosum is completely absent in 57.8% of patients with the disease, partially absent in 9.4% of patients, and normally present in 32.8% of patients [Muscular Dystrophy Canada, 2013].
Worldwide prevalence of the disease is extremely low. Four cases have been reported in other countries: 2 in Italy, and 2 in Turkey. In the population of Saguenay–Lac-Saint-Jean, the prevalence is 1 in 2,117 births, while 1 in 23 individuals is a carrier of the recessive gene [Dupré et al., 2014].
HMSN/ACC is caused by a mutation in the gene SLC12A6 (c.2436delG), in exon 18 on chromosome 15. In one case, the mutation c.1584_1585delCTinsG was reported on exon 11 of the same chromosome. In 2002, researchers from Quebec isolated the c.2436delG mutation—occurring in more than 99% of cases—on exon 18 [Dupré et al., 2003].
Clinical manifestations of HMSN/ACC occur shortly after birth or during the first year of life and include muscular weakness and delayed motor development (difficulty holding head up, sitting, standing, and walking). The disease is progressive, leading to a loss of mobility and to malformations of the vertebral column, hands and feet. Mild to moderate delayed intellectual development is observed in patients requiring special education as of grade 2 or 3. The diagnosis is based on the identification of genetic mutations, on clinical manifestations, neurological examinations, brain imaging (MRI, CT scans) and EMG [Dupré et al., 2014].
There is no known cure for the disease. Children usually begin to walk at around the age of 3.8 years and stop walking at around the age of 13.8 years; they often need corrective surgery for thoracic deformities as well as physiotherapy and a significant amount of support, since they gradually lose their autonomy [Muscular Dystrophy Canada, 2013]. The mean age at death among people with hereditary motor and sensory neuropathy with or without agenesis of the corpus callosum is 33 years [Dupré et al., 2014].
3.2.4 Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset progressive neurological disorder that mainly affects people from the Charlevoix and Saguenay–Lac-Saint-Jean regions, as well as people whose ancestors are from these areas.
ARSACS is a neurodegenerative disease characterized by early-onset cerebellar ataxia with spasticity, a pyramidal syndrome, and peripheral neuropathy (Table 1). The disease manifests between the age of 12 and 18 months, with patients having gait disturbance and walking difficulties [Fontaine, 2008]. Other early signs of cerebellar ataxia include dysarthria (difficulty pronouncing words) and nystagmus. School-age children have difficulty carrying out manual activities and writing, and problems with sports such as bicycling, skiing or skating [Muscular Dystrophy Canada, 2007]. During adolescence, teenagers will face
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increasing difficulty walking and performing activities requiring manual dexterity; therefore, young adults will often require mobility aids such as a cane and then a wheelchair by around age 30 [Dystrophie musculaire Canada, 2007].
The worldwide prevalence of ARSACS is unknown, but similar cases have been reported in Turkey, Japan, Holland, Italy, Belgium, France, and Spain [Fontaine, 2008]. However, the age of onset in these cases is more variable and older than in Quebec. In the Charlevoix-Saguenay region, the incidence at birth is estimated at 1 in 1,932, while 1 in 22 individuals is a carrier of the gene [Fontaine, 2008; Mercier et al., 2001].
ARSACS is caused by mutations in the SACS gene, which is located on chromosome 13 (13q11) and encodes a large protein called sacsin; the specific function of the protein is yet unknown (Table 1).
The structure of sacsin has recently been described [Kozlov et al., 2011]. Another discovery showed that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with another protein in mitochondrial fission. Consequently, mitochondrial dysfunction has been identified as the likely cellular basis for ARSACS [Girard et al., 2012]. Two mutations were reported in families of the Charlevoix-Saguenay region: c.6594delT (96% of cases) and c.5254C>T (in two families) [Mercier et al., 2001]. It should be noted that with the growing number of ARSACS cases worldwide, at least a dozen other mutations have been discovered, which may explain the high variability in clinical expression and severity of the disease when it is reported outside Quebec [Thiffault et al., 2013].
The clinical diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay is generally established at a young age and is based on the results of neuroimaging studies (MRI, CT scans) and neurophysiological data. Retinal examinations show that hypermyelination of the retinal nerve fibers (without loss of vision) is a constant feature in patients with the disease [Fontaine, 2008].
The disease progresses slowly, and the life expectancy of people with the disease is 60 to 70 years. Treatment must be scalable and adapted to the different needs of patients and the progression of their condition: among other things, this includes preventing spasticity from a young age, physiotherapy, pharmacotherapy, and the use of ankle-foot orthoses [Fontaine, 2008; Muscular Dystrophy Canada, 2007].
3.3 Number of Patients Targeted
The screening test is intended for adults aged 18 years and over who are planning to have children, who live in Saguenay–Lac-Saint-Jean, or who have a parent from this region or from the Charlevoix or Haute-Côte-Nord region. The test is also available to expecting couples at less than 14 weeks’ gestation.
In Quebec, an estimated 7,000 people per year will undergo a screening test, 5,000 of whom will be from Saguenay–Lac-Saint-Jean.
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3.4 Medical Specialties and Other Professions Involved
All health professionals involved in prenatal care who work with expecting couples, such as prenatal nurses, obstetricians (family physicians and specialists), pediatricians, and genetic counsellors, must be involved in the screening process.
Family physicians, pediatricians and health professionals specializing in genetic screening should also be involved in the screening of children and adults.
3.5 Testing Procedure
According to the standard operating procedure (SOP), blood samples are collected on blotting paper from the Réseau de médecine génétique appliquée du Québec (RMGA).
4 TECHNOLOGY BACKGROUND
4.1 Nature of the Diagnostic Technology
This test is not listed in the Index, but it has been part of a genetic screening pilot project sponsored by the MSSS and ASSS 02 since 2009.
4.2 Brief Description of the Current Technological Context
The simultaneous screening of four genetic diseases of autosomal recessive inheritance is carried out using TaqMan® PCR technology (Appendix A).
4.3 Brief Description of the Advantages Cited for the New Technology
The technology has several advantages: it is very simple to use, accurate, and rapid. Moreover, the weighted value is reduced by five units per test compared with the former multiplex technology panels.
4.4 Cost of Technology and Options: Not assessed.
5 EVIDENCE
5.1 Clinical Relevance
5.1.1 Other Tests Replaced
A new test was introduced following a pilot study undertaken in March 2009. The multiplex panel on Luminex platform developed for the pilot project was recently replaced by TaqMan® quantitative PCR technology (Life Technologies).
This technology has several advantages: it is specific, fast, simple to use, less costly .
5.1.2 Diagnostic or Prognostic Value
The screening test is specific to four autosomal recessive diseases and will allow carrier status for one (or several) of these diseases to be established. Regardless of the disease, in these specific regions, carrier status is found in 1 out of 20 to 1 out of 23 people, while 1 in approximately 2,000 children is affected.
Each of these four diseases is severe, debilitating and has a very poor prognosis in terms of morbidity and mortality.
Identification of carrier status will help provide adequate genetic counselling, either before pregnancy or during the first trimester.
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5.1.3 Therapeutic Value: Not applicable.
5.2 Clinical Validity
COMPONENT PRESENCE ABSENCE NOT APPLICABLE
Sensitivity X
Specificity X
Positive predictive value (PPV) X
Negative predictive value (NPV) X
Likelihood ratio (LR) X
ROC curve X
Accuracy
Sensibility (Se) and Specificity (Sp)
To assess sensitivity and specificity, a large number of carriers of a mutation must be tested, which can be challenging in the case of rare mutations. As indicated in the October 19, 2009, progress report, "results for 92 carriers had to be validated without any inconsistencies to be 95% certain that the sensitivity and specificity is greater than or equal to 0.95." For the most common mutations, 84 (lactic acidosis) to 106 carriers were identified (more than 92 for tyrosinemia, polyneuropathy and the first mutation in ARSACS) (Table 2).
As no divergent results were found, "the sensitivity and specificity can be assessed with 100% confidence for tyrosinemia, polyneuropathy and the first mutation in ARSACS; as well, sensitivity and specificity arevery high for lactic acidosis and the second mutation causing ARSACS" (Final report, May 17, 2013; unpublished paper).
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Table 2: Assessment of the pilot project for four inherited recessive diseases of Saguenay–Lac-Saint-Jean
YEAR SAMPLES SERIES TECHNIQUE CONCORDANCE SE SP AD HOC RETESTING
COMPLETE RETESTING
TURNAROUND 5-10-20 DAYS
1
2009 1,755 (n = 351)
27 Multiplex on Luminex platform
100% (-1)2 < n
3 < n 10% > 50% 7 to 13
13 n. a.
2010 - - Multiplex 100% < n < n - 3 periods: 13%, 27% and 7%
-
2013 (total) (03/09-11/12)
2,866 86 Multiplex 100% 100%4 100%
4 5% 12.8% 5
5.7 5.9
2014 2,400 - TaqMan® 100% - - 0.4% 1.0% -
Abbreviations: n = number of patients who were tested for the five mutations of the four diseases; n. a. = not available; Se = sensitivity; Sp = specificity. 1 turnaround = time between receiving the blotting paper at the laboratory and validating the results by the medical team in Quebec based on an 1) "urgent" (5 days) (prenatal sample: 13 -16 weeks); 2) "stat" (10 days) (prenatal sample < 13 weeks); and 3) "routine" (20 days) (preconception sample) status. 2 Concordance: Over a 2-year period, results obtained in the local laboratory were compared with those obtained at the CHUQ and CHU Sainte-Justine (10 samples sent and received, including at least one carrier of each mutation and one noncarrier (-1: rare polymorphism). 3 < n: insufficient sample size. 4 Sensitivity and specificity of 100% for tyrosinemia, polyneuropathy and the first mutation in spastic ataxia (since a sufficient number of carriers, at least 92, were detected; in fact, 106 carriers of these 3 mutations were identified). Only 84 carriers were identified for lactic acidosis and 16 for the second mutation of spastic ataxia, which indicates "very high" sensitivity and specificity, according to the May 17, 2013 final report.
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The authors caution that "the performance of the multiplex test is probably comparable to that of conventional technologies, since no inconsistencies were noted to date. However, an estimate of the sensitivity and specificity of conventional technologies was not published, which does not allow an accurate comparison to be made of the performance of the various analytical approaches" (May 17, 2013, final report).
It should be noted that the clinical validation was conducted with the help of three medical biochemists from the CHUQ.
5.3 Analytical (or Technical) Validity
COMPONENT PRESENCE ABSENCE NOT APPLICABLE
Repeatability X
Reproducibility X
Analytical sensitivity X
Analytical specificity X
Matrix effect X
Correlation between test and comparator X
Other, depending on type of test X
The screening of four autosomal recessive diseases of Saguenay–Lac-Saint-Jean was part of a pilot project that ran from March 2009 to November 30, 2012. The technology consisted of a multiplex panel on a Luminex platform. Therefore, the clinical validation was carried out mainly with this method. As certain technical difficulties led to a high rate of complete retesting, TaqMan® hydrolysis probe-based real-time PCR was adopted towards the end of 2012 and in 2013. The assessment of the technical validation with one method will therefore be reported more briefly than with the other.
Standard Operating Procedures
The locally developed screening test was meticulously planned, following external performance audits and a report from the Comité d’éthique de santé publique (pilot project providing carrier tests, internal documentation of the pilot project, 2006–2013). Two progress reports (2009–2010) and a final report (2013) provide the results of the development, performance, and technical validations of these tests. Standard operating procedures (SOP), which were last updated on September 23, 2013, were developed and implemented to ensure adequate quality control.
Internal Quality Control
Internal quality control is applied to the reagents, sampling, and analysis of control and sample results. For each series of tests, there is at least one negative control (DNA replaced with water), a non-carrier control, a heterozygous carrier and, if possible, a homozygous carrier.
For each new primer and probe test kit, the analysis of a 16-sample series will include at least 4 known carriers and a negative reference sample.
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External Quality Control
External quality control implemented at the beginning of the pilot project was complied with. Therefore, on 6 occasions, or every 3 months over a period of 21 months, 10 samples were sent to the CHU Ste-Justine and the CHUQ, while the CSSS de Chicoutimi analyzed 10 samples from each of the hospitals. The 10 samples included at least one carrier for each of the 5 mutations and at least one non-carrier. Therefore, in 21 months, 280 samples were sent and the CSSS de Chicoutimi received 160 samples. Concordance was perfect at 100% (discordance: 0%) (Table 2).
External Validity
External validity was carried out in two situations:
1) when both spouses were carriers of a mutation in the same gene, which occurred 19 times and was confirmed in 100% of cases by the two other hospitals (Final report, 2013);
2) in the case of atypical fluorescence. There were 55 cases of atypical fluorescence for the p.A354V mutation in the gene responsible for lactic acidosis that were caused by a polymorphism (rs4953042) located a few nucleotides downstream from the site of the mutation.
This was determined with TaqMan® technology developed in Chicoutimi (final report, 2013). The results of the multiplex PCR test also were inconclusive in 2 out of 79 cases. These cases were sent to the reference laboratories for validation, since it is impossible to obtain consistent fluorescence patterns for a mutation with multiplex technology. The new TaqMan® technology should remedy this problem (Final report, 2013).
Results Turnarounds
Compliance with turnarounds based on specimen status was another laboratory performance measure. It refers to the period between receiving the blotting paper at the laboratory and validating the results by the medical team in Quebec, based on an "urgent" (prenatal specimen 13 to 16 weeks), "stat" (prenatal specimen < 13 weeks), or "routine" (preconception specimen) status. The turnaround times were set at 5, 10 and 20 days, respectively.
After 6 months, the turnaround time for the "urgent" status was 7 to 13 days, rather than 5 days, and that for the "stat" status was 13 days rather than the 10 days anticipated. The deadlines were extended as a result of technical problems that required the complete retesting of a series of tests (Progress report 1, 2009; Table 2).
Nevertheless, the problem appeared to be resolved towards the end of the pilot project, when all the turnaround times were approximately 5 days, regardless of sample status (Final report, 2013; Table 2).
Retesting
The first report of 2009 found that 10% of the sample set required retesting for a participant (ad hoc retesting), which meets the objectives set at the beginning of the pilot project. The complete retesting of entire series of tests was more extensive than initially anticipated, and in 60% of cases, was attributable to non-compliant results of negative controls. Of the tests carried out with the PCR method, 10 had to be repeated, and of those performed with the Luminex platform, 5 had to be repeated (Progress report 1, 2009).
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The second report of 2010 again shows high and inconsistent rates of complete retesting, varying between 13%, 27% and 7.3% after several modifications (Table 2, Preliminary report II, 2010).
The final, cumulative report (2013) states that there were 143 ad hoc retests of the 2,886 samples, representing a rate of 5%, whereas 11 of the 86 series over the 2 years of the pilot project had to be completely retested (frequency of 12.8%) (Table 2, Final report, 2013).
The high rate of complete retesting led to the development of a replacement methodology towards the end of 2012. Since then, almost 2,400 samples have been assessed using TaqMan® technology, and the resulting retesting frequency was found to be exceptionally low: 0.4% for ad hoc retesting and 1% for complete retesting (letter dated February 5, 2014; Table 2).
TaqMan® Technology
TaqMan® technology was developed concurrently by the requester, and on May 17, 2013, it was validated at more than 75% (Final report, 2013). This technology has been reported to be already in place at the CHU Ste-Justine.
According to the requester, the technology presents several advantages: There is no need for DNA extraction; the amplification cycle is short (< 40 min); the results can be read during the test; there is no need to manipulate the PCR products (lower risk of contamination); and only one reagent has to be purchased (in addition to primers and probes), which reduces quality control requirements (Final report, 2013).
Moreover, TaqMan® technology is considered to be more robust, accurate, rapid, and less costly, and it can perform a greater number of tests simultaneously than other technologies.
5.4 Recommendations from Other Organizations
This section is not applicable, as this notice addresses inherited recessive diseases specific to the Saguenay–Lac-Saint-Jean and Charlevoix regions regarding ARSACS.
6 ANTICIPATED OUTCOMES OF INTRODUCING THE TEST
6.1 Impact on Material and Human Resources
Well-trained personnel are already present. However, the number of tests might increase to 7,000 per year, from approximately 3,000 tests performed during the 2 years of the pilot project.
6.2 Economic Consequences of Introducing Test Into Quebec's Health Care and Social Services System
Not assessed.
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6.3 Main Organizational, Ethical, and Other (Social, Legal, Political) Issues
The availability of routine screening in the region will need to be accompanied by more extensive genetic counselling resources. Ethical issues were addressed in the July 2007 notice issued by the Comité d’éthique de santé publique.
7 IN BRIEF
7.1 Clinical Relevance
This involves a new screening test developed and assessed through a pilot project (March 2009 to November 2012). The test establishes carrier status for one or more of four inherited autosomal recessive disorders specific to the Saguenay–Lac-Saint-Jean or Charlevoix region. Carrier status is found in 1 out of 20 to 23 people, while 1 out of 2,000 children is born with one of these diseases. All diseases targeted in the screening test (lactic acidosis, tyrosinemia type I of Saguenay–Lac-Saint-Jean, HMSN/ACC and ARSACS) are severe and debilitating and have a very poor prognosis in terms of morbidity and mortality.
7.2 Clinical Validity
Although the locally developed technology was not compared with other technologies, its sensitivity and specificity are essentially 100%. It should be noted that the sensitivity and specificity of the proposed TaqMan® technology were not reported. However, given the accuracy and robustness of the method, it is expected to perform as well.
7.3 Analytical Validity
The quality control measures were implemented in accordance with the standard operating procedures developed by the requester. The internal quality controls have been described. There is complete agreement (100%) between the tests conducted in the requester's laboratory and the tests conducted in two other university laboratories in Quebec. However, if only one establishment is designated to perform the tests, external validation procedures should be implemented for the new technology to ensure quality control, particularly if both members of the couple are carriers of the mutation.
Turnaround times for analysis of test results were met, particularly following the introduction of TaqMan® technology, which considerably reduced the frequency of ad hoc or complete retesting. It should be noted that the technical validity of the TaqMan® method for these tests appears to be only 75% complete. While the new TaqMan® technology is very robust, simple to use, accurate, and specific to these analyses, the technical validation for these four diseases of Saguenay–Lac-Saint-Jean needs to be completed and the rate of ad hoc and complete retesting needs to be kept to a minimum.
7.4 Recommendations from Other Organizations: Not applicable.
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8 INESSS NOTICE IN BRIEF
Four Diseases of Saguenay–Lac-Saint-Jean – Mutations Screening
Status of the Diagnostic Technology
Established, but not validated for the mutations under study
Innovative
Experimental (for research purposes only)
Replacement for technology which becomes obsolete
INESSS Recommendation
Include test in the Index (see additional comments)
Do not include test in the Index
Reassess test when the validation with the new TaqMan technology is completed
Additional Recommendation
Draw connection with listing of drugs, if companion test
Produce an optimal use manual
Identify indicators, when monitoring is required
Notes
It is necessary to regulate testing procedures and to ensure that quality control, including external validation, is maintained.
Testing five mutations simultaneously may generate irrelevant results in screened populations; it is important to ensure that testing requirements for these mutations are set out separately.
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REFERENCES
Agence d’évaluation des technologies et des modes d’interventions en santé (AETMIS). Spectrométrie de masse en tandem et dépistage néonatal des erreurs innées du métabolisme – Rapport technique. Rapport préparé par Héla Makni, Carole St-Hilaire, Laura Robb, Kathy Larouche et Ingeborg Blancquaert. Montréal, Qc : AETMIS; 2007.
Bergeron A, D’Astous M, Timm DE, Tanguay RM. Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type I. J Biol Chem 2001;276(18):15225-31.
De Kok JB, Wiegerinck ETG, Giesendorf AJ, Swinkels DW. Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes) Hum Mutat 2002;19(5):554-9.
De Laet C, Dionisi-Vici C, Leonard JV, McKiernan P, Mitchell G, Monti L, et al. Recommandations for the management of tyrosinemia type I. Orphanet J Rare Dis 2013;8:8.
De Lonlay P. Tyrosinemiatype 1 [site Web]. Orphanet; 2007. Available at: http://www.orpha.net/consor/cgi-bin/OC_Exp.php?Lng=FR&Expert=882.
Dupré N, Howard HC, Rouleau GA. Hereditary motor and sensory neuropathy with agenesis of the corpus callosum. Dans : Pagon RA, Adam MP, Ardinger HH, et al., réd. GeneReviews® [Internet]. Seattle, WA : University of Washington; 2014. Available at: http://www.ncbi.nlm.nih.gov/books/NBK1372/.
Dupré N, Howard HC, Mathieu J, Karpati G, Vanasse M, Bouchard JP, et al. Hereditary motor and sensory neuropathy with agenesis of the corpus callosum. Ann Neurol 2003;54(1):9-18.
Dystrophie musculaire Canada. Neuropathie sensitivo-motrice héréditaire [site Web]. Toronto, ON : Dystrophie musculaire Canada; 2013. Available at: http://muscle.ca/la-dystrophie-musculaire/les-maladies-neuromusculaires/neuropathie-sensitivo-motrice-hereditaire/ (consulté le 9 avril 2014).
Dystrophie musculaire Canada. L’ataxie de Charlevoix-Saguenay (ARSACS). Toronto, ON : Dystrophie musculaire Canada; 2007. Available at: http://muscle.ca/wp-content/uploads/2012/11/435F_L_ataxie_de_Charlevoix_Saguenay_f.pdf (consulté le 9 avril 2014).
Fontaine B. Ataxie spastique autosomique récessive de Charlevoix-Saguenay [site Web]. Orphanet; 2008. Available at: http://www.orpha.net/consor/cgi-bin/OC_Exp.php?Lng=FR&Expert=98.
Girard M, Larivière R, Parfitt DA, Deane EC, Gaudet R, Nossova N, et al. Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). Proc Natl Acad Sci 2012;109(5):1661-6.
Grompe M. Disorders of tyrosine metabolism. Waltham, MA : Wolters Kluwer Health; 2014. Available at: http://www.uptodate.com.
Kozlov G, Denisov AY, Girard M, Dicaire MJ, Hamlin J, McPherson PS, et al. Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). J Biol Chem 2011;286(23):20407-12.
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Life Technologies. TaqMan® SNP genotyping assays. Product Bulletin. Grand Island, NY : Life Technologies; 2011. Available at: http://tools.lifetechnologies.com/content/sfs/brochures/cms_040597.pdf.
Mercier J, Prévost C, Engert JC, Bouchard JP, Mathieu J, Richter A. Rapid detection of the sacsin mutations causing autosomal recessive spastic ataxia of Charlevoix-Saguenay. Genet Test 2001;5(3):255-9.
Morin C. Acidose lactique congénitale type Saguenay-Lac-Saint-Jean [site Web]. Orphanet; 2011. Available at: http://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=FR&Expert=70472.
Orphanet. Le portail des maladies rares et des médicaments orphelins [site Web]. Paris, France : Orphanet. Available at: http://www.orpha.net/consor4.01/www/cgi-bin/?lng=FR (consulté le 26 mars 2014 pour les 4 maladies).
Ouellette G. Trousse d’information sur lactic acidosis. À l’intention des parents d’enfants atteints de lactic acidosis. Jonquière, Qc : Association de lactic acidosis du Saguenay-Lac-Saint-Jean; 2008. Available at: http://www.aal.qc.ca/File/Trousse_Information-AAL.pdf.
Thiffault I, Dicaire MJ, Tetreault M, Huang KN, Demers-Lamarche J, Duquette A, et al. Diversity of ARSACS mutations in French-Canadians. Can J Neurol Sci 2013;40(1):61-6.
Other internal documents provided by the requester.
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APPENDIX A TaqMan® Assays
PCR with Hydrolysis Probe (or TaqMan® Assay [trademarked by Life Technologies/Applied Biosystems])
The assay consists in amplifying the region(s) containing one or more single nucleotide polymorphism (SNP) to be tested with oligonucleotide primers and characterizing the SNP using specific probes. These probes contain a fluorochrome at their 5′ end (such as FAM, VIC, etc.) and a fluorescence inhibitor (quencher) at the 3′ end, which inhibits fluorescence in intact probes. For genotyping, a semi-quantitative measurement of fluorescence is performed at the end of the PCR (unlike real-time assays, which are quantitative and assess fluorescence at each cycle). The number of probes included in the assay depends on the possible SNPs. There are often two probes: one that hybridizes with the normal allele and one that hybridizes with the mutant allele. Each type of probe contains a fluorochrome that distinguishes it from the other. Several TaqMan® assays are available on the market. They are used for frequently sought mutations such as C282Y or H63D in the HFE gene (in hemochromatosis). However, it is possible to order synthesis of probes and primers independently (or have an assay custom-made).
During the PCR reaction, the denaturation, annealing and elongation steps unfold as they would in ordinary PCR, with the difference that during the annealing step, not only do the primers hybridize with the DNA template, but also the probe that is perfectly complementary to the sequence. For the elongation step, TaqMan technology will synthesize a strand complementary to the template using a primer. As the primer meets the probe and extends, it degrades the probe (5′ exonuclease activity), causing it to release probe-specific fluorochrome, which will then emit fluorescence as a result of its sudden separation from the quencher at the 3’ end. The type of fluorescence and its semi-quantification at the end of PCR with a real-time thermal cycler will allow the patient's genotype (homozygous normal or mutant or heterozygous) to be determined.
(Summary by Nathalie Girard, Ph. D.)
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Figure 1: Diagram of the basic principle of genotyping with hydrolysis probes
Taken from Life Technologies. TaqMan® SNP genotyping assays. Product Bulletin. 2011. Available at: http://tools.lifetechnologies.com/content/sfs/brochures/cms_040597.pdf.
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Figure 2: Example of detection and genotype assignment during an assay
Taken from Life Technologies. TaqMan® SNP genotyping Assays. Product Bulletin. Available at: http://tools.lifetechnologies.com/content/sfs/brochures/cms_040597.pdf.
VIC fluorescence alone was detected; this indicates that the probe hybridized perfectly to the mutant allele = homozygous mutant genotype
FAM fluorescence alone was detected; this indicates that the probe hybridized perfectly to the normal allele = homozygous normal genotype
The 2 fluorescence signals were detected = heterozygous genotype
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ANNEXE B Details Concerning the Genes and Technology
Five mutations sought for the following four diseases:
Congenital lactic acidosis (LRPPRC p.A354V);
Hereditary tyrosinemia type I (FAH IVS12+5G>A);
Hereditary motor and sensory neuropathy with or without agenesis of the corpus callosum (SLC12A6 2436delG);
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (SACS 6594delT and 5254C>T).
Amplification of the gene regions containing the five mutations targeted by this test is carried out using the polymerase chain reaction technique. An amplification reaction is required for each mutation. The amplicons generated by PCR amplification are detected with specific TaqMan® probes (Life Technologies) at each of the 10 possible alleles. Once the amplification is completed, the samples are analyzed using an ABI 7500Fast (Life Technologies) real-time thermal cycler, which measures the fluorescence emitted by each of the ten probes. Whether or not an amplicon is hybridized to each of the ten probes determines the presence or absence of fluorescence. The test is based on Watson and Crick's base-pairing principle and is highly specific. The principle is widely used in molecular genetics.
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APPENDIX C Abbreviations
AA Amino acid
CLA SLSJ Congenital lactic acidosis, Saguenay–Lac-Saint-Jean type
ARSACS Autosomal recessive spastic ataxia of Charlevoix-Saguenay
COX Cytochrome-c oxidase enzyme
FAH Fumarylacetoacetate hydrolase (final enzyme in the tyrosine breakdown pathway)
HT1 Hereditary tyrosinemia, type 1
LRPPRC Leucine-rich pentatricopeptide repeat motif-containing protein (involved in the stabilization and transport of mature mitochondrial RNA)
LSJ Lac-Saint-Jean
HMSN/ACC Hereditary motor and sensory neuropathy with agenesis of the corpus callosum. This disease causes the corpus callosum to be completely absent in 57.8% of patients, partially absent in 9.4%, and present in 32.8% (Muscular Dystrophy Canada, April 9, 2014)
