Supplemental Figures of Phenotypes linked to duplication upstream of SOX9: New insights into presentation and diagnosis

Supplemental Figure 1. Microarray analysis images of family 2 and 3. Patient IV.1 of family 2 is the index 46,XX/SRY-negative case (A). Increased logR ratio (the top graph) and altered heterozygozity pattern of B-allele frequency, BAF (the bottom graph) in 17q24.3 chromosomal region indicates heterozygous duplication in 5’upstream regulatory region of the SOX9 gene. Asymptomatic 46,XX mother who carries SOX9 upstream duplication (B). Asymptomatic 46,XX sister who carries SOX9 upstream duplication (C). Patient V.4 of family 3 is the index 46,XX/SRY-negative case (D). Asymptomatic 46,XX mother who carries SOX9 upstream duplication (E). Microrray testing of family 2 was performed using Infinium Global Screening Array-24+V3.0 kit (~700K) and analyses were performed on NxClinical program. Microrray testing of family 3 was performed using Infinium HumanCytoSNP-850K V2.1 kit and analyses were performed on BlueFuse Multi v4.5 software.

Supplemental Figure 2. Appearance of the external and internal genitalia and histological structure of the gonads of patient II.1 with 46,XX/SRY-negative ovotesticular DSD from family 4. Asymmetric external genitalia with a single palpable gonad in the right labioscrotal fold and a small phallus (A). Laparoscopic examination of the internal genital organs revealed a well-developed testis in the right labioscrotal region (indicated by black arrow) (B) and a macroscopically normal ovary and tuba in the pelvis on the left side (indicated by black arrow) (C). However, there was no uterus or sex cords identified in the pelvic area. Histological examination of the gonads revealed normal immature testis with seminiferous tubules containing Leydig and Sertoli cells in the right gonad (D), and normal ovarian tissues with follicles and stroma in the left gonad (E).

Supplemental Figure 3. Scanned pictures of multiplex ligation-dependent probe amplification (MLPA) analysis of patient II.1 and his father (I.2) from family 4. Patient II.1 who has 46,XX/SRY-negative ovotesticular DSD shows increased signal (duplication) in SOX9 probe (indicated by black arrow; heterozygous duplication in the region of probes 301-373 in the SOX9 upstream region) and absent SRY probe signal in MLPA (A). Asymptomatic father (I.2) who carries SOX9 duplication (indicated by black arrow) has the similar image for SOX9 probe (indicated by black arrow) and normalSRY probe signal in MLPA image (B). MLPA analysis was performed using MRC Holland SALSA^® MLPA^® Probemix P185 Intersex kit (https://www.mrcholland.com/product/P185/2052).