Molecular Analyses
For family NMD02, fluorescently labeled microsatellite markers located
close to the respective genes (Table S1) were genotyped to exclude
linkage of the disorder to GNPTAB (OMIM#607840), GNPTG(OMIM#607838), IDUA (OMIM#611542) and ARSB(OMIM#611542). Samples from two affected individuals III:5 and III:9
from family NMD02 were chosen for exome sequencing using the Agilent
human V5 kit (51Mb), (Agilent Technologies, Santa Clara, CA, USA).
Paired-end reads were obtained at 50X average target coverage on an
Illumina Hi-Seq 2500 sequencer (Otogenetics, Norcross, GA, USA). Samples
from parents and the affected child were assessed by exome sequencing
for family ID01.
For family NMD02, we analyzed the exome data for identification of
variants unique to each patient, as well as for those identical to both
affected individuals. Variants in the known genes for Mucolipidosis and
Mucopolysaccharidosis (Table S1) were also checked. All homozygous
variants and compound heterozygous variants in the exome data were
examined further after filtering. Variants with allele frequencies of
less than 0.01 in all public databases were retained. Further choice was
made for exonic missense, nonsense frameshift and in-frame insertion or
deletion variants. Synonymous and intronic variants affecting splice
sites or creating new splice sites, were also considered.
Exome variant call files for two affected individuals of NMD02 were
analyzed with Agilevariant as described (Carr et al., 2013) to display
regions of homozygosity in individual data and those common to both
samples were identified (Supplementary Figure 1). Exome coverage data
were examined to identify incompletely sequenced exons in one or both of
the samples. Those exon located within regions of shared homozygosity
were sequenced after amplification with specific primers (Table S2)
using a standard PCR protocol or a protocol for GC-rich
amplification.(Naz & Fatima, 2013)
Data for the three samples of family ID01 was analyzed by Burrow Wheel
Aligner (BWA) (Li & Durbin, 2009), GATK (McKenna et al., 2010) and
ANNOVAR.(Wang, Li, & Hakonarson, 2010)