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)