Xi Yang, Akihiro Hoshino, Takashi Taga, Tomoaki Kunitsu, Yuhachi Ikeda, Takahiro Yasumi

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Supplementary materials for " A female patient with incomplete hemophagocytic lymphohistiocytosis caused by a heterozygous XIAP mutation associated with non-random X-chromosome inactivation skewed towards the wild-type XIAP allele " in Journal of Clinical Immunology

Xi Yang, Akihiro Hoshino, Takashi Taga, Tomoaki Kunitsu, Yuhachi Ikeda, Takahiro Yasumi, Kenichi Yoshida, Taizo Wada, Kunio Miyake, Takeo Kubota, Yusuke Okuno, Hideki Muramatsu, Yuichi Adachi, Satoru Miyano, Seishi Ogawa, Seiji Kojima, Hirokazu Kanegane

Corresponding author

Hirokazu Kanegane MD, PhD

Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sicences, Tokyo Medical and Dental University, Tokyo, Japan

E-mail:

Supplemental Figure 1. Pedigree of the family.

The female patinet (Patient 3) is indicated by arrow.

Supplemental Figure 2. cDNA sequence analysis of XIAP gene in the family.

cDNA sequence was detected by antisense analysis. Upper panel indicates wild type, and the second, third and forth panels indicate Patients 1, 2 and 3, and lower panel indicates their mother. The arrow indicates the mutation site (insertion C).

Supplemental Figure 3. XIAP expression in lymphocytes from the patients and normal control by western blot.

XIAP expression was detected from PBMC of the patients and normal control using the clone 48 antibody (upper panel), the clone 2F1 antibody (middle panel) and the b-actin antibody as the internal control (lower panel). Patients 1, 2 and 3 showed deficient expression of XIAP protein.

Supplemental Figure 4. XCI pattern in PBMCs from the father, mother and Patient 3.

DNA was extracted from PBMCs of the father mother and P7atient 3 and amplified by PCR with specific primer that flank the exon 1 of the androgen receptor. In addition, the DNA was digested with the methylation-sensitive enzyme HpaII enzyme (New EnglandBiolabs, Beverly, MA) before PCR amplification. The samples were analyzed on SDS polyacrylamidegel(PAGEL AE-6000; ATTO, Tokyo, Japan) and stained with SYBR-Gold (Invitrogen, Carlsbad, CA). Minus (-) and plus (+) mean no digestion and digestion by HpaII enzyme.

Supplemental Figure 5. X-chromosome inactivation pattern in hair root cells from the mother and Patient 3.

Patient 3 showed an extremely skewed X-chromosome inactivation (XCI) pattern with 100:0, in which the maternally inherited mutated X-chromosome is exclusively activated and the paternally inherited normal X-chromosome is exclusively inactivated, whereas the mother showed a random XCI pattern with 59:41 in hair root cell.

Supplemental Figure 6. AICD analysis of the family members and 6 normal controls.

The PBMC were stimulated phytohemagglutinin and rIL-2. After 7 dyas of culture, activation-induced cell death (AICD) assays were performed, and percentages of apoptotic cells were evaluted using propidium iodide-negative and Annexin-V-positive staining by flow cytometry.