Patient 1 was a male neonate (gestational age 38 weeks and 6 days) born by cesarean section. Apgar scores at 1, 5, and 10 minutes were 10. The patient weighed 2180 g at birth, head circumference was 34 cm, and birth length was 47 cm. The birth was considered ‘low birth weight’ (BW<2500 g) and ‘intrauterine growth restriction’ (weight < 10th percentile); therefore, the infant was admitted to a neonatal intensive care unit. Physical examination showed the infant’s neck was slightly shorter and the muscle tension of the limbs was slightly lower than normal; however, there were no other clinically significant changes in the skeletal muscle at this time. A myocardial enzyme test revealed high levels of creatine kinase (2317.00 U/L; normal 50-310 U/L), creatine kinase isoenzyme (59.00 U/L; normal 0-17 U/L), and lactate dehydrogenase (579.0U/L; normal 120-250U/L). Cardiac ultrasound examination demonstrated a patent foramen ovale but no cardiac hypertrophy. MRI examination of the brain was unremarkable. At 2 months of age, the infant’s cervical vertebrae caused his head to be skewed to the right (Figure 1A), and muscle tension of the limbs was slightly lower than normal. During follow-up, the infant’s growth and development were delayed. At 7 months of age, the infant’s weight was < 10th percentile, body length was on the 3rd percentile, and head circumference was approaching the 10th percentile, having been on the 25th percentile after the birth (Table 1). The infant failed to meet developmental milestones as he could not sit alone at 7 months of age.
Patient 2 was a 32 year old male and the father of Patient 1. Patient 2 developed scoliosis and kyphosis during childhood (the male of Fig. 1 B or Fig. 1C), but there were no clinical signs typical of LDM in adulthood, such as flexor muscle weakness, calf hypertrophy, or the inability to lift the first toe. The patient’s height (153 cm) was shorter than normal, and he had an atypical gait. Cardiac ultrasonography showed no ventricular hypertrophy.
Patient 3 was Patient 2’s mother. Patient 3 had the same phenotype as Patient1 and 2, including progressive scoliosis, a hunchback, and difficulty walking during childhood(the female of Fig. 1C). Patient 3 did not consent to genetic testing. Information on other family members was not available.
Whole genome sequencing
Whole genome sequencing was performed using the second-generation high-throughput method. 2 ml peripheral venous blood was sampled from each patient into an ethylenediaminetetraacetic acid (EDTA) anticoagulant tube. Genomic DNA was extracted using the QIAamp DNA Extraction Kit (QIAGEN), according to the manufacturer’s instructions. DNA concentration was estimated by measuring the absorbance at 260 nm. DNA was extracted using magnetic beads. PCR amplification was performed followed by ligation with specific linkers. Gene regions were captured with two TruSight One Sequencing Panels (illumina Inc, USA) and amplified by PCR. The exons of 4811 clinically relevant genes were sequenced using a MiSeq sequencer (illumina Inc, USA).
Data were aligned on the UCSC hg19 reference sequence using the BWA algorithm (8) and annotated with published literature (9). Pathogenicity of mutations was predicted using clinical data and bioinformatics software (PolyPhen2, LRT, Mutation Taster). Potential candidate mutations were obtained by analyzing the function, variation, and inheritance pattern of each gene.
Candidate mutation sequences were validated by Sanger sequencing. Primer sequences for PCR were Sense: 5'-GACCAGATGAATGAGCACCG-3'; Antisense:5'-AGGGCGTTCTTCGCCTTAAC-3'. After obtaining informed consent, whole exome sequencing was performed on the patients to identify novel variants residing in the MYH7 gene.