A 32-month-old boy was admitted to our hospital with two days of irritability and fever. He was born with a birth weight of 2.9 kg by normal spontaneous vaginal delivery at 40 weeks gestation to a healthy 28-yr-old G3P2 mother. His delivery was non-eventful, but there was flexion deformity of both his middle fingers, so he was transferred to our hospital and underwent many diagnostic tests and physical therapies in January, 2002. He showed mild general hypotonia in that his cry was weak, and he sucked poorly during early infancy. He had epileptic seizures 6 times since the age of 15 months despite taking anti-epileptic medications. A diagnosis of mental retardation complicated by epilepsy was made, and his development was noted to be markedly delayed. He raised his head at age 7 months, sat up at 26 months, and had not yet acquired the ability to stand. He had a flat and mid-hypoplastic face with prognathism, narrow and upward slanting palpebral fissures with hypertelorism, low-set ears, a small crashed nose, widely spaced incisors, carp-like mouth, and round back (Fig. 1). He demonstrated repeated stereotyped behavior like hitting his chin with his palm; this behavior was often associated with emotional outbursts.
Soon after admission, a generalized tonic-clonic type seizure was started and lasted approximately one hour; at that time his vital signs were as follows: blood pressure 100/60 mmHg, pulse rate 138 bpm, respiration rate 32 bpm, and body temperature 38.5C. Mild throat injection was seen, but other systemic examinations were normal. On neurologic exam, mental status was alert, all cranial nerve exams were normal, and muscle tone in both extremities were decreased to grade IV/IV bilaterally but all deep tendon reflexes were physiologic. There was no spasticity or pathologic reflex. Sensory functions were normal and meningeal irritation signs were absent.
Initial laboratory investigations revealed: hemoglobin 12.7g/dL, platelet count 374,000/microL and white blood cell count 11,780/microL. The C-reactive protein was slightly elevated at 19.0 mg/dL on the day of admission and returned to normal 3 days later. Urinalysis was normal. All microbiologic studies were negative. A sleep electroencephalogram revealed intermittent high-amplitude slow wave discharges from the temporo-occipital area, representing mild cerebral dysfunction. On radiologic evaluation, spine radiography revealed mild scoliosis. An echocardiogram was normal.
The characteristic facial features and mental retardation of the patient led us to consider ATR-X syndrome; thus, peripheral RBCs were screened for hemoglobin H (HbH) inclusion bodies. HbH inclusions were detected under the microscope in 1.1% of brilliant cresyl blue stained RBCs, consistent with the diagnosis of ATR-X syndrome (Fig. 2). The patient's father, mother and two sisters were all given the same test; only his mother and eldest sister had the same findings. We performed gene analysis to confirm the diagnosis of ATR-X syndrome. Genomic DNA from the peripheral blood of our patient and his 4 family members was extracted with the Wizard  Genomic DNA Purification Kit (Promega, Madison, WI, USA) and used as a template for amplification of the ATRX gene. Polymerase chain reaction (PCR) was separated into three parts and carried out with sequence-specific oligonucleotide primers containing 20 known major mutation sites (Table 1, Figs. 3, 4). PCR amplification using each primer was conducted with premixture kit (PreMix -Top, Bioneer Inc., Seoul, Korea). Cycling conditions were one cycle at 95C for 5 min, followed by 35 cycles of 95C for 30 sec, at an annealing temperature adjusted to each primer's (Tm) for 30 sec, 72C for 30 sec, followed by one cycle at 72C for 7 min. For direct sequencing, the PCR products were purified using a PCR clean-up system offered by Promega. DNA sequencing was performed using the purified PCR products as template, the same primers as those used for template generation, the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA), and automatic sequencer ABI Prism 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence data were assembled and compared to that of a previously reported ATRX sequence from accession number Z84487 using the DNAstar software (DNASTAR Inc., Madison, WI, USA).
Mutation analysis for our patient showed a point mutation on the 9th exon in the ATRX gene of thymine to cytosine so that Trp(C), the 220th amino acid, was replaced by Ser(R) (Fig. 5). This missense mutation was reported by Gibbons in 1997. We investigated the same mutation in his 4 family members, and his mother and two sisters were found to be carriers.