Genomic DNA and total RNA isolation from leukocytes and urine sediment
Quick Gene Mini 80 system (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and Quick-DNA Urine kit (Zymo Research Corporation, Irvine, CA, USA) were used to extract genomic DNA from peripheral blood leukocytes and urine sediment of patient. The obtained genomic DNA was used for targeted sequencing, Sanger sequencing, and HUMARA assay.
Total RNA of patient’s blood leukocytes was extracted by A Ribopure Blood Kit (Invitrogen, Carlsbad, CA, USA) by adding RNA stabilization agent (RNAlater; Invitrogen) in to the whole blood. ZR Urine RNA Isolation Kit (Zymo Research Corporation) was used for RNA extraction from urine sediment. Ultra-deep targeted RNA sequencing was performed by using both extracted total RNA.
Detection of pathogenic variants of GLA
To detect the pathogenic variant in GLA, we conducted comprehensive analysis by next-generation sequencing (NGS) using MiSeq (Illumina, San Diego, CA, USA) for all patients. The sample library for NGS analysis was prepared using HaloPlex Target Enrichment Kit 500 kb for Illumina (Agilent Technologies, Santa Clara, CA, USA), in accordance with the manufacturer’s workflow. In brief, for aech sample to be sequenced, an individual target enriched, index library is prepared by digest and denature the sample DNA, hybridize probe library to DNA targets, ligate and capture uniquely barcoded targets and amplified fragments by PCR. All indexed DNA samples were amplified by polymerase chain reaction and sequenced using the MiSeq platform. Variant calling was performed by using a SureCall 4.0 software (Agilent Technologies).
Variant that were called by SureCall were confirmed using Sanger sequencing. Primer pairs were constructed to amplify mutational site of GLA in each patient. After 40 cycles of amplification, PCR products were separated on 1.5% agarose gel and subjected to direct sequencing using dye terminator cycle sequencing kit (Amersham Biosciences, Piscataway, NJ, USA) and an automatic DNA sequencer (ABI Prism 3130; PerkinElmer, Applied Biosystems, Foster City, CA, USA). For variant description, NM_000169 was used as a reference sequence.
The HUMARA assay was performed as described by Allen et al. . We used 200 ng of genomic DNA from blood leukocytes and urine sediment to be digested by a methylation-sensitive enzyme (Hpa2). Digested and undigested DNA was amplified with FAM-labeled forward primer and reverse primer specific for regions either side of polymorphic CAG repeats (forward primer: 5-TCCAGAATCTGTTCCAGAGCGTGC3 ; reverse primer: 5-GCTGTGAAGGTTGCTGTTCCTCAT-3). The product was amplified and mixed with internal size standard (GeneScan 500 LIZ Dye Size Standard; PerkinElmer, Applied Biosystems). Quantification of data and their visualization in graphs were performed using GeneScan software. The XCI pattern was defined as random (from 50:50 to 80:20), skewed (from 80:20 to 90:10), or extremely skewed (more than 90:10) .
Ultra-deep targeted RNA sequencing using NGS
We recently developed a novel assay of ultra-deep RNA sequencing for examining XCI patterns  that uses the quantification of transcript expression for wild-type and variant alleles. Total RNA from blood leukocytes and urine were reverse transcript by using Ecodry Premix (Double Primed; Clontech Laboratories Inc., Mountain View, CA, USA). To ensure the purity of the RNA, we amplified the cDNA by glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific primers, which were designed to amplify both genomic DNA and cDNA to confirm genomic DNA contamination (forward primer: 5-CCCTTCATTGACCCTCAAC-3, reverse primer: 5-TTCACACCCATGACGAAC-3). Nested RT-PCR was then performed using forward and reverse primers designed according to the mutation site. The cDNA PCR product was purified on 1.5% agarose gel, the ends of the cDNA fragments were repaired and adenyl nucleotides were added using TruSeq Nano DNA Library Prep for Illumina (Santa Clara, CA, USA) following the Haloplex or SureSelect NGS Target Enrichment Workflow (Agilent Technologies) in the NGS analysis. The results were analyzed using SureCall 3.0 (Agilent Technologies). We analyzed the ratio of variant to wild-type alleles for estimation of the XCI .