Testis development in the absence of SRY: chromosomal rearrangements at SOX9
and SOX3 - SUPPLEMENTARY MATERIAL
Annalisa Vetro; Mohammad Reza Dehghani; Lilia Kraoua; Roberto Giorda; Silvana
Beri; Laura Cardarelli; Maurizio Merico; Emmanouil Manolakos; Alexis Parada
Bustamante; Andrea Castro; Orietta Radi; Giovanna Camerino; Alfredo Brusco; Marjan
Sabaghian; Crystalena Sofocleous; Francesca Forzano; Pietro Palumbo; Orazio
Palumbo; Savino Calvano; Leopoldo Zelante; Paola Grammatico; Sabrina Giglio;
Mohamed Basly; Myriam Chaabouni; Massimo Carella; Gianni Russo; Maria Clara
Bonaglia, Orsetta Zuffardi
Corresponding author: Prof. Orsetta Zuffardi, Dept. Molecular Medicine, University
of Pavia, via Forlanini 14, 27100 Pavia, Italy; Tel. +390382987733; E-mail:
zuffardi@unipv.it
Supplementary Materials and Methods
SRY analysis
The presence of SRY gene sequence was excluded by PCR (cases 1-3 and all the cases
reported in supplementary table 1) or FISH (Fluorescence in-situ hybridization) analysis
(case 20). For PCR detection, amplification was performed under standard conditions.
Primers are available upon request. FISH analysis was performed by using commercial
probes (Cytocell Ltd, Cambridge, United Kingdom).
Molecular karyotyping
Molecular karyotyping was performed for cases 1-3 and for all cases reported in
supplementary table 1 by using oligonucleotide array-CGH platforms (180K SurePrint
G3 Human Kit, Agilent Technologies, Santa Clara, CA), as reported elsewhere.1 A
46,XX reference DNA (NA15510, Coriell Cell repositories) was used for all
experiments. Changes in DNA copy number at a specific locus were observed as the
deviation of the log2ratio value from 0 of at least three consecutive probes, by using
Genomic Workbench v. 5.0.14 software (Agilent, ADM-2 algorithm with a threshold of
5). Oligomer positions refer to the Human Genome GRCh37 (hg19) assembly. Copy
number variations reported in the Database of Genomic Variants
(http://projects.tcag.ca/variation/) were excluded from further analysis. Confirmation of
the results and segregation analysis were performed for cases 1 and 2 by qPCR as
reported elsewhere.2 For case 20, microarray analysis of the trio was performed using
the Genome Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA) as previously
described.3 Genotyping and copy number analysis were performed with the Genotyping
Console Software 4.1 (Affymetrix, Santa Clara, CA, USA) using annotation file version
NA32 (hg19) and an in-house reference file consisting of 90 samples (45 males and 45
females). A minimum cut-off value of 25 consecutive probes with a consistently
aberrant signal was included in our criteria to ascertain a copy number change.
Translocation breakpoint mapping (case 3)
Based on case 3 cytogenetics data, we selected ten roughly evenly-spaced primer pairs
(11-1 to 11-10) from non-repeated portions of a 100-kb region on chromosome 11p13
(35,920,000 to 36,020,000) and seven pairs (17-1 to 17-7) on a 50-kb region on
chromosome 17q24.3 (69,160,000 to 69,220,000). Average distance between
consecutive primers on the same strand was 10 kb; average distance between
plus(F)/minus(R) strand primers in each pair was 500 bases. We set up long-range PCR
reactions with each chromosome 11 plus-strand primer and a pool of chromosome 17
plus-strand primers, and each chromosome 11 minus-strand primer and a pool of
chromosome 17 minus-strand primers on case 3 DNA and normal control DNAs.
Primer 11-2R selectively amplified from the proband a 2-kb fragment, restricting the
chromosome 11 breakpoint to the 10-kb region between 11-1 and 11-2 primers. Next,
each chromosome 17 primer was tested separately, and the chromosome 17 breakpoint
was restricted to the 7-kb region between primers 17-4 and 17-3. The der11 breakpoint
junction was amplified with primers 11-2R and 17-4R. Additional primers (11-BPF and
17-BPF) were selected to amplify the der17 breakpoint junction. Primer sequences are
available on request.
Supplementary Figure Legend
Supplementary Figure 1 – Hematoxylin and eosin-stained section of the gonadal
biopsy from case 2 showing both testicular and ovarian tissue. On the left a
seminiferous tubule is indicated by the larger arrow; the ovarian follicles are indicated
on the right by the two smaller arrows.
Case n.
case_4
case_5
case_6
case_7
case_8
case_9
case_10
case_11
case_12
case_13
case_14
Age
at
Ascertain
the
Genitalia
ment
diag
nosis
infertility,
adult male with
27 azoosperm
hypoplastic testes
ia
28
Other clinical
findings
Laboratory Findings
short stature
increased FSH, LH and
androstenedione, low
serum testosterone
infertility,
adult male with
azoosperm
hypoplastic testes
ia
ambiguous
at
external
birth
genitalia
ambiguous
at
external
birth
genitalia
negative at mutation
analysis for RSPO1
hypoplastic
uterus,
abdominally
retained gonads,
ovotestis
low serum testosterone
hypospadia, right
testis palpable in
the scrotum, left
testis visible by
ultrasound
Δ4-androstendione 5.4
nmol/L, 17-OH
progesterone >60,5
mol/L, cortizole/serum
594, testosterone 14,7
pmol/L, DHEAS 8,3
µmol/L, E2/serum 2,65
nmol/L
Other
Galliera Genetic
Bank, funded by
Telethon Italy
provided this
sample
(01GGB13711M13577)
infertility,
adult male with
azoosperm
hypoplastic testes
ia
ambiguous
ambiguous
TOP
external
external genitalia
genitalia
hypospadias,
ambiguous
at
micropenis and
external
birth
bilateral inguinal
genitalia
cryptorchidism
28
16
yrs
hypogona
dism
male with
hypoplastic testes
hypospadias,
micropenis and
ambiguous bilateral inguinal
at
external
cryptorchidism;
birth
genitalia left gonadectomy
at 16 years of age,
ovotestis
hypertrophy of
clitoris, single
urethral meatus,
ambiguous
at
urogenital sinus,
external
birth
abdominally
genitalia
retained gonads
low responsive to
HCG, ovotestis
at
ambiguous enlarged clitoris
failure to thrive
from 7 yrs,
short stature
low GH; Increased
FSH, normal LH and
testosterone (17 yrs)
bilateral
gynecomastia
elevated 17β-estradiol
and LH, low serum
testosterone
increased FSH, normal
Clinical description
reported by ParadaBustamante et al,
2010 4
birth
case_15
case_16
case_17
case_18a
– Family
1*
case_18b
– Family
1*
case_19a
– Family
2§
case_19b
– Family
2§
external
genitalia
resembling a
penis, ovotestis,
absent uetrus
hypoplastic testes
ambiguous
at
and prostatic
external
birth
hypoplasia at
genitalia
ultrasound
ambiguous
at
ambiguous
external
birth
external genitalia
genitalia
ambiguous
at
ambiguous
external
birth
external genitalia
genitalia
bilateral
ovotestis, small
ambiguous
hypospadic
at
external
phallus,
birth
genitalia
rudimentary
uterus, tubes,
vaginal atresia
small hypospadic
ambiguous
at
phallus, absent
external
birth
uterus, sight tests,
genitalia
left ovotestis,
hypertrophy of
clitoris
resembling a
ambiguous
at
penis, urogenital
external
birth
sinus, bifid and
genitalia
empty scrotum,
hypoplastic
uterus, ovotestis
ambiguous hypertrophy of
external
clitoris, empty
at
genitalia; labioscrotal folds,
birth hypertroph
abdominally
y of
retained gonads,
clitoris
ovotestis
LH, testosterone:
230ng/dl (male value)
increased FSH and LH
Clinical description
reported by
Fraccaro et al, 1979
gynecomastia
5
Clinical description
reported by
Fraccaro et al, 1979
gynecomastia,
stature <3rd
centile
5
hyperthyroidis
m
increased testosterone
pre-term birth
(32 wks)
increased testosterone
gravida 2 para 1
Supplementary Table 1 – Summary of clinical information for 18 cases of 46,XX SRY
negative individuals with DSD for whom genomic arrays did not highlight any
significant CNV.
*Cases 18a and 18b belong to family 1 and are two siblings previously described by
Fraccaro et al, 1979.5
§Cases 19a and 19b are a mother and her daughter (family 2), both with ovotestis.
TOP: induced termination of a pregnancy
Supplementary References
1.
Vetro A, Manolakos E, Petersen MB et al: Unexpected results in the constitution
of small supernumerary marker chromosomes. European journal of medical
genetics 2012; 55: 185-190.
2.
Vetro A, Ciccone R, Giorda R et al: XX males SRY negative: a confirmed cause
of infertility. J Med Genet 2011; 48: 710-712.
3.
Palumbo O, Palumbo P, Palladino T et al: An emerging phenotype of interstitial
15q25.2 microdeletions: clinical report and review. American journal of medical
genetics Part A 2012; 158A: 3182-3189.
4.
Parada-Bustamante A, Rios R, Ebensperger M, Lardone MC, Piottante A, Castro
A: 46,XX/SRY-negative true hermaphrodite. Fertility and sterility 2010; 94:
2330 e2313-2336.
5.
Fraccaro M, Tiepolo L, Zuffardi O et al: Familial XX true hermaphroditism and
the H-Y antigen. Human genetics 1979; 48: 45-52.