Spinal Muscular Atrophy (SMA) Biomarkers Study in the Immediate Postnatal Period of Development

• SMA Molecular Biomarker: SMN mRNA Levels [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]

  SMN mRNA determination:

  Quantitative RT-PCR: Absolute quantification of full length SMN transcripts will be performed essentially as described (Tiziano, Pinto et al. 2010). Briefly, SMN1 and SMN2 transcripts will be measured in a multiplex reaction and SMN-del7 will be quantified separately. Quantification of GAPDH transcripts will serve to control for RNA quantity and reverse transcriptase efficiency. No template and no reverse transcriptase controls will be included to track potential DNA contamination.

  Multiplex digital PCR: Droplet digital PCR (ddPCR) results in an absolute measure of target molecules. It does not rely on measurements in the log phase of PCR but instead measures the absolute count of positive gene copies. ddPCR will be used to determine SMN levels to further increase reliability and reduce variance in the SMN mRNA level determination.

  
  
• SMA Biomarker: SMN protein levels (SMN protein in PBMCs) [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]

  SMN protein in PBMCs SMN cell-based immunoassay: Cell immunoassays will be performed(Kolb, Gubitz et al. 2006). This assay is performed using a single monoclonal antibody for SMN and does not involve the disruption of cells. The sensitivity and reliability of this assay will be compared to the commercially available ELISA assay.

  SMN ELISA: From the same PBMC sample, a commercially available SMN ELISA will also be performed according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY). It is possible that insufficient blood volumes will be collected at any given visit. The analysis will be prioritized as follows from the CPT tube: 1) SMN protein level using cell-based immunoassay, 2) SMN protein level using SMN ELISA. These samples will be coded, and the parent/guardian may request at any time that the child's samples be destroyed at this processing laboratory.

  
  
• SMA Biomarker: Panel of plasma molecules (SMAF SMA Signature Panel) [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]
  SMAF SMA Signature Panel The B for SMA pilot study identified over 100 protein analytes that significantly correlated with motor function (as measured by Modified Hammersmith Functional Motor Scale) in SMA patients compared to controls (http://neuinfo.org/bforsma). The SMAF is currently contracted with Myriad Rules-Based Medicine (RBM) to develop a test for a subset of these proteins. The significance of these proteins is uncertain. For example, the proteins that most correlated with motor function in the B for SMA study was cartilage intermediate layer protein 2 and cartilage oligomeric matrix protein. This test analysis will be administered by the company Myriad RBM. These samples will be coded, and the parent/guardian may request at any time that the child's samples be destroyed at Myriad RMB.
  
  

• SMA Physiological Biomarker: Electrical Impedance Myography (EIM) [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]
  Convergence Medical Devices, Inc. has developed a handheld device specifically for performing EIM measurements on patients of all ages including infants. Data obtained with this handheld EIM device has shown marked alterations in the data between healthy and sick patients including those neurogenic and myopathic diseases. The device has not been used in infants to date. However, it has undergone independent safety testing by Intertek and has been used in more than 200 individuals, including 92 pediatric subjects ages 3-12, without any adverse events (AEs). EIM non-invasively measures the impedance of skeletal muscle. We anticipate that EIM will thus likely be able to assist in quantifying the severity of the disease affecting various muscle groups as well as in measuring changes in the disease over time. EIM measurements will be conducted on a core set of four muscles: biceps, the wrist extensor compartment, quadriceps, and tibialis anterior.
  
  
• SMA Physiological Biomarker: Compound Motor Action Potential Testing [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]

  Maximumulnar CMAP amplitude and area will be obtained by recording from the abductor digitiminimi muscle following ulnar nerve stimulation at the wrist. All electrophysiologic testing will be performed by certified electromyographers experienced in the assessment of pediatric patients. Maximum values for both negative peak (NP) amplitude and NP area will be obtained. No medications will be used.

  This test is done routinely in this population. Pediatric electrodes and each site's standard electromyograph devices will be utilized. The test, while not considered to be painful, may cause some discomfort similar to a static electric shock. Infants may whimper or cry due to the surprise of the shock. If the first test does not provide adequate results, the evaluator may do a quick second or third stimulus to confirm the maximal response. Each shock lasts approximately 0.1 millisecond. The testing duration is expected to be approximately 30 seconds.

  
  
• Motor Function Scale: Test for Infant Motor Performance Screening Items (TIMPSI) [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]
  Test for Infant Motor Performance Screening Items (TIMPSI): The TIMPSI is a reliable and validated, comprehensive assessment of the postural and selective control of movement needed by infants less than 5 months of age for functional activity in the early months of life (Morton, MacLaren et al. 2006; Campbell, Swanlund et al. 2008; Finkel, Hynan et al. 2008). This assessment includes up to 29 different items that assess the child's motor abilities. The test will be performed by a trained pediatric physical therapist. The detailed instructions on administration of the test are provided in the manual of operations. Reliability and validity of the TIMPSI for infants with SMA Type 1 has recently been established.
  
  
• Motor Function Scales: Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND): [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]
  Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP IN-TEND): The CHOP INTEND was designed to evaluate the motor skills of patients with SMA-1.(Glanzman, Mazzone et al. 2010) The CHOP INTEND is a reliable measure of motor skills in patients with SMA-I and neuromuscular disorders presenting in infancy. However, further vali-dation is needed. The detailed instructions on administration of the test are provided in the ma-nual of operations.
  
  
• Motor Function Scale: Alberta Infant Motor Scale (AIMS): [ Time Frame: Up to 24 months ] [ Designated as safety issue: No ]
  Alberta Infant Motor Scale (AIMS): The AIMS incorporates the neuromaturational concept and the dynamical systems theory and is used to measure gross motor maturation of infants from birth through the age of independent walking (Piper, Pinnell et al. 1992, Piper, Darrah et al 1994). In the AIMS, the impact of neurological components on motor development is reflected by a sequence of motor skills, which are used as the basis of assessment. (Piper, Darrah et al. 1994). The scale follows the principles of dynamical systems because motor skills are tested by observing infants as they move into and out of 4 positions: prone, supine, sitting, and standing. The detailed instructions on administration of the test are provided in the manual of operations.
  
  

October 2013 Enrollment Update: The healthy control enrollment has been filled and is now closed. The study is still enrolling eligible infants with SMA.

Aim 1. To establish the validity of putative physiological SMA biomarkers in the immediate postnatal period. A longitudinal, natural history examination of physiological markers of muscle innervation will be performed in healthy and SMA infants. The first week of life is the ideal first time point, with visits occurring at scheduled visits up to the age two. Compound motor action potential (CMAP) amplitude and electrical impedance myography (EIM) will be examined and will be correlated with motor function. Each of these is associated with muscle innervation and provides information on the number and function of lower motor neurons in the spinal cord, the cellular target of SMA therapeutic interventions. This trial will establish the natural history of these putative SMA biomarkers as the disease evolves in affected infants. Moreover, our approach will allow for measurements in pre-symptomatic and early symptomatic subjects and determine their predictive value.

Aim 2. To establish the validity of putative molecular SMA biomarkers in the immediate postnatal period.SMN2 copy number is a valid, predictive molecular SMA biomarker; however, it is fixed, and therefore not useful as a biomarker of clinical progression or response to therapy. SMN mRNA and protein expression is variable in different cell types and, in mice, naturally decreases with age postnatally. In this study, SMN expression levels will be measured longitudinally in SMA patients and controls. Additional putative molecular SMA markers that have been identified to correlate with motor function will be determined in an effort to distinguish between predictive markers that change prior to development of weakness and those that change as a consequence of weakness.