Contributed by Faye F. Gao MD, PhD and Marie C. DeFrances MD, PhD
An eight year old boy with a history of autism and cerebral palsy was brought to his pediatrician by his mother for new onset staring episodes. According to the mother, the infrequent episodes are typified by the boy stopping suddenly, staring and becoming unresponsive. The mother also noted that the child has been sleeping more in recent weeks. The child's history is negative for convulsions, myoclonic jerks, meningitis, encephalitis or severe head trauma associated with loss of consciousness.
PATIENT BIRTH HISTORY
The pregnancy was complicated by gestational diabetes in the mother who was treated with insulin. She also had elevated liver function tests (LFTs) during pregnancy; otherwise, no additional problems were noted.
The patient was born at 38 weeks via C-section. He was 7 pounds 14 ounces. The patient was diagnosed with neonatal jaundice, treated with phototherapy and discharged three days after birth.
PAST MEDICAL HISTORY
The mother and her sister are bipolar. The mother is adopted; therefore, extended family history is limited. To the mother's recollection, though, no one in the family has a history of seizures. However, the maternal grandmother and maternal uncle are mentally retarded. The maternal uncle also has large, low set ears. The patient has a healthy 10 year-old sister and his father's health condition is unknown. See family tree (Fig. 1).
PHYSICAL EXAMINATION AND MEDICAL WORK-UP
The patient's physical examination is positive only for dysmorphic features with large, low set ears. An EEG was performed and showed a normal awake-and-asleep pattern; however, a subtle slow background frequency for age was noted. An MRI was positive for a thin corpus callosum and mildly increased signal in periatrial white matter suggesting previous white matter insult. A blood sample from the patient was also sent for DNA molecular testing.
MOLECULAR TEST RESULTS
Southern blot analysis was performed to assess the patient's FMR-1 gene to determine whether the patient's CGG repeat range falls into the normal, permutation or full mutation categories. DNA was extracted from the patient's peripheral blood leukocytes and digested with specific enzymes, followed by agarose gel electrophoresis to separate the digested DNA by size. The gel was transferred to nitrocellulose and hybridized to a radiolabeled probe specific to FMR-1. In Fig. 2A, a Southern blot result in which the patient's DNA was digested with the restriction enzyme PstI, a methylation insensitive restriction enzyme, is shown. Digestion with PstI reveals a 1 kb band in normal individuals and larger band(s) in those individuals with premutation or full mutation FMR-1 alleles. In our patient's sample, Pst1 digestion showed several bands substantially >1 kb ranging in size ranging from 2 to 2.7 kb in Southern blot. The dominant 2.7 kb band was estimated to contain 530 CGG repeats. In Fig. 2B, the patient's DNA was digested by a combination of EagI and EcoRI. EagI and EcoRI are two restriction enzymes widely used in Southern blot to assess the methylation status of the FMR-1 allele(s). EagI is a methylation sensitive enzyme while EcoRI is not. EagI and EcoRI used in combination reveal a 5.2 kb band which is the methylated inactive FMR-1 allele seen in normal females and a 2.8 kb band representing the unmethylated active allele in normal males and females. In individuals with fully expanded FMR-1 alleles, one or more bands >5.2 kb in size representing methylated alleles can be seen (Fig. 3). In our patient, multiple bands ranging in size from 6.1 to 9.2kb representing methylated FMR-1 alleles were observed. A 4.1 kb band was also identified which may represent a partially methylated FMR-1 allele. No normal unmethylated allele was observed for our patient (Fig. 2B). Together the data are consistent with the patient having a fully expanded, fully methylated FMR-1 allele with evidence of mosaicism (Fig. 2A and B).