EXPRESSION OF CFTR mRNA IN NASAL EPITHELIUM AND VAS DEFERENS · University of Toronto The gene...

EXPRESSION OF CFTR mRNA

IN NASAL EPITHELIUM

AND VAS DEFERENS

Victor Mak, M.D.

A thesis submitted in conformity with

the requirements for the degree of

Master of Science

Graduate Department of Institute of Medical Science

University of Toronto

O Copyright by Victor Mak 1997

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University of Toronto

The gene responsible for cystic fibrosis (CF), called the cystic fibrosis

transmembrane conductance regulator (CFTR), encodes the CAMP-regulated chloride

channel found in the apical membrane of secretory epithelial cells. It has been well

established that almost al1 males with CF are azoospermie due to atrophy or absence of

structures derived fiom the Wolffian duct. Interestingly, a higher than expected

fiequency of mutations in the CFTR gene has been identified in men with congenital

absence of vas deferens and men with epididymal obstruction. In particular, these

individuals have been found to have a significantly higher incidence of the 5-thymidine

(5T) variant of the CFTR intron 8 polypyrimidine tract (IVS8-T tract) compared to

normal or CF populations. The 5T variant results in less efficient splicing of CFTR exon

9 compared to the more common 7T and 9T variants and therefore produces less normal,

full-length CFTR mRNA. The protein produced by the CFTR transcript lacking exon 9

fails to fùnction as a CAMP-dependent chloride channel. The fact that these infertile

males have no other clinical signs of classical CF suggests that the epithelia of the male

reproductive tract may have the highest requirement for CFTR fûnction or, altematively,

splicing of CFTR mRNA in the reproductive tract is less efficient than the other CF-

associated organs. Nasal epithelia and segments of vas deferens were obtained fiom 24

healthy, previously vasectomized men who presented for vasectomy reversal.

Quantitative RT-PCR was performed on these specimens, with the region of CFTR

cDNA spanning exon 9 amplified. For both nasal and vasal tissues, a strong positive

correlation was found between the length of the TVS8-T tract and the proportion of

&A with exon 9 intact. In addition, within the same subject, a significantly higher

level of transcripts lacking exon 9 was found in vas deferens than nasal epithelia,

regardless of the NS8-T genotype. These findings suggest that the splicing of CFTR

precursor mRNA is less efficient in vasal epithelia compared to respiratory epitheiia.

Thus, differential splicing efficiency between the various tissues which express CFTR

provides one possible explanation for the reproductive tract abnormalities observed in

infertile men with CFTR gene alterations but without other manifestations of CF.

The author wishes to express sincere appreciation

to Prof. Lap-Chee Tsui, Dr. Keith Jarvi, and

Dr. Johanna Rornmens for their assistance in the

preparation of this manuscript.

.......................................................................................................................... List of Tables iv

.......................................................................................................................... List of Figures v

Abbreviations .......................................................................................................................... vi

.............................................................................................. Attribution of Labor and Data 1

1 . Introduction ...................................................................................................................... 2

............................................................................................................ I.A. Background 2

....................................................................................... 1 .A. 1 . Human Male Znfirtility 2

I.A. 1 .a. Overview of Male Testicular and

.............................................................. Reproductive Tract Embryology 2

.................................................................... I.A. 1 .ai. Development of testes 2

............................................... I.A. 1 .aii . Development of male genital ducts 3

........................................ I.A. 1 .aii i. Development of male external genitalia 4

I.A.1 . b. OveMew of Male Reproductive Physiology .............................................. 4

.......................................... I.A. 1 . b.i. Hypothdamic-pituitary-testicular axis 5

........................................................................................ 1.A.l .b.i i. The testis 5 .. ......................................................................... LA.1 .b.ii. a. Leydig cells 5

.......................................................... I.A. 1 .b.ii. b. Seminiferous tubules 6 . . I.A. 1 .b.ii. c. Spermatogenesis ................................................................ -7

......................................... I.A. 1 .b.ii i. Hormonal control of spermatogenesis 7

.................................... I.A. 1 .b.i v. Sperm maturation, storage, and transport 8

LA . 1 .b. v. Fertilization ................................................................................... -9 . . .................................*.......,................... LA 1 c. Clinical Aspects of Male Infertility 9

................................................... I.A. 1 .c .i. Evaluation of the infertile male 10

................................................................. I.A. l .c.i. a. Semen analysis 1 0

................................... l.A.l .c.i i. Etiologic classification of male infertility 11

l.A.l . c i i. Treatment of male infertility .................................................... 11

Conductance Regulator ................................................................................. 1 2

I.A.3. Cystic Fibrosis Trunsmembrane Conductance Regulator and

........................................................................ Mesonephric Duct Anomalies 13

I.A.4. The Intron 8 Polypyrimidine Tract of t he CFTR Gene and

Alternative Splicing of CFTR Exon 9 ....................................................... 1 8

I.A.5 . The 5T Variant in Iniron 8 of the CFTR Gene and

Congenital Absence of Yas Deferens ............................................................. 19

I.B. Hypothesis ........................................................................................................... 21

1.C . Purpose ................................................................................................................ 21

.................................................................................................. II . Materials and Methods 22

Patients and samples ........................................................................................... 22

Allele specific oligonucleotide analysis of NS8-T tract ................................... 22

................................. PCR amplification of CFTR exon 9+ and exon 9- cDNA 24

Analysis of CFTR mRNA by reverse transcriptase-

polymerase chain ration .............................................................................. 25

Quantification of CFTR mRNA transcripts ....................................................... 27

Analysis of data ................................................................................................... 27

............................................................................................................................. III . Resuits 29

m.A. WS8-T tract genotype ......................................................................................... 29

iII.B. Conversion of experimental to actual proportions of

................................................................................... exon 9+ CFTR mRNA 29

ïiI.C. Evaluation of exon 9+ and exon 9- CFTR mRNA transcripts .......................... 31 ........................................................... m.D. Quantification of exon 9+ CFTR mRNA 32

m.E. Relationship between level of exon 9+ C F ' mRNA

....................................................................................... and the IVSS-T tract 32

iIi.F. Cornparison of the proportion of exon Pt CFTR mRNA

in nasal epitheliurn to vas deferens ................................................................ 32

- . - - - - - - - - - - - - -

1V.A. Splicing efficiency of CFTR exon 9 is related to

the iVS8-T tract length ...... .. . ......... ............ ..... . .. .. ... ...... , .. . . .. .. ............. . . . ...... .. .33

N . B . Higher proportion of normal CFTR mRNA in

nasal epithelium than vas deferens ................................................................ 33

W.C. The CFTR IVS8-T tract modifies phenotype by variable

splicing of exon 9 in different tissues ............................................................ 34

1V.D. Proportion of normal CFTR mRNA produced by

the WS8-T aileles ........ .... ................... . ... ..... ..... ....... .... ...........,..... . ................. 3 5

W.E. Molecular mechanisrn of differential splicing efficiency

of CFTR exon 9 .............................................,................................................ 37

N . F . Summary ............................................................................................... . ............. 38

V. Future Directions .... . .......... .... ........ .., . .. . ,..... .. ........... ....., .. ...... . ..... . . . . .... ...... ....... . . . . ...... .. -40

VI. References .. . . ... . . . . ... . . . . . . . . . . .. . . . . . . . .. . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . ... -42

Table 1. World Health Organization criteria for

normal semen analysis .................................................................................. 54

Table 2. CFïR gene mutations or variations and CBAVD ............................................ 55

Table 3. Frequencies of the WS8-T alleles in subjects from

present study and in general population ..................................................... 56

Table 3. Proportion of Cl?T'R exon 9+ mRNA in

nasal epithelia and vas deferens ................................................................... 57

Table 4. Estimated proportion of CFTR exon 9+ transcripts

produced by the IVS8-T tract alieles ........................................................... 58

Table 5. Estimated proportion of CFTR exon 9+ transcripts

produced by the WS8-T tract genotypes .................................................... 59

Figure 1. The intron 8 polypyrimidine tract of the CFTR gene

and alternative splicing of CFTR exon 9 .................................................... 60

Figure 2. Detection of IVSS-T genotype by

oligonucIeotide hybridization ........................ .. ..... .. ..... ... ..... .................. . ...... -62

Figure 3. Determination of proportion of exon 91 CFTR mRNA .................................. 65

Figure 4. Evaluation of RNA extraction and cDNA synthesis ........................................ 72

Figure 5. Evaluation of CFTR mRNA ......................................... ............ ... ....................... 74

Figure 6. Quantification of nasal and vas deferens CFTR mRNA ................................ 76

Figure 7. Relationship between CFTR IVSS-T genotype

and proportion of exon 9+ transcripts in

nasal epithelia or vas deferens. ....... ....... ............... ..................................... ... 78

Figure 8. Cornparison of the proportion of CFTR exon 9+

transcripts within nasal epitheiia and

vas deferens from the same subject ...... .. .. .. .. . . .... .. . . ... . . . .... . . ......... ...... ..... .. ... 80

Figure 9. Influence of CFïR genotype on phenotype by

the proportion of normal CFTR mRNA .................................................. 82

ABC

ATP

ARTS

bp

CAMP

CAVD

CBAVD

cDNA

CF

CF-PI

CF-PS

CFTR

CUAVD

DHT

DNA

ciNTP

EDTA

FSH

GAPDH

GnRH

rvF

adenosine triphosphate binding cassette

adenosine triphosphate

assisted reproductive technologies

base pair(s)

cyclic adenosine monophosphate

congenitai absence of vas deferens

congenital bilateral absence of vas deferens

complementary deoxyribonucleic acid

cystic fibrosis

pancreatic-insufficient cystic fibrosis

pancreatic-sufficient cystic fibrosis

cystic fibrosis trammembrane conductance regulator

congenital unilateral absence of vas deferens

dihydrotestosterone

deoxyribonucleic acid

deoxynucleoside triphosphate

ethylenediamine tetraacetic acid

follicle-stirnulating hormone

glyceraldehyde-3-phosphate dehydrogenase

gonadotropin-releasing hormone

in vifro fertilization

rvs

LH

M

MESA

P8

MIE:

min

cl1

ml

PM

mM

mRNA

*g

OD

PCR

RNA

RNase

RT

RT-PCR

S

SD

SDS

intervening sequence (intron)

luteinizing hormone

mole(s)/liter

microsurgical epididymal sperm aspiration

micrograrn(s)

Mullerian inhibitory factor

minute(s)

microliter(s)

milliliter(s)

micromole(s)/liter

millimole(s)/liter

messenger ribonucleic acid

nanogram(s)

optical density

polymerase chah reaction

ribonucleic acid

ri bonuclease

reverse transcriptase

reverse transcriptase-polymerase chah reaction

second(s)

standard deviation

sodium dodecyl sulfate

vii

SSC

TAE

TDF

TdT

TSH

VS.

x2

sodium citrate/sodiurn chloride

Tris-acetate, EDTA buffer

testis determining factor

terminal deoxy transferase

thyroid-stimulating hormone

versus

chi-square

viii

Mrs. Theresa Longley served as study nurse for the project and assisted in specimen

collection, patient counseling, and obtaining patient consent.

Dr. Keith Jarvi fiom the Division of Urology, Department of Surgery, Mount Sinai

Hospital, Toronto provided tissue specimens.

My contribution to this work includes the following:

collection of specirnens

obtaining patient consent

extraction of DNA fiom pet-ipheral leukocytes

genotyping of CFTR IVS8-T tract

extraction of RNA

quantitative RT-PCR experiments

determination of proportion of exon 9+ CFTR transcnpts

design of experimental protocols

9. analysis and interpretation of data

I.A. BACKGROUND

I.A.1. HUMAN MALE INFERTILITY

Approximately fifteen percent of couples are infertile [Mosher, 19851. An

abnormality is found in only the man in 30 percent of the cases, while abnomalities

detected in both partners occur in another 20 percent. Therefore, male factor infertility

accounts for one half of couples with fertility problems [Mosher, 1985; Sigman and

Howards, 19921. Infection, immunologie factors, congenital anomalies, genetic defects,

toxic insult, and sexual dystunction contribute to this important medicai problem. In this

section, the embryology of the male gonad and reproductive tract, the physiology of male

reproduction, and clinical aspects of male infertility will be reviewed.

1.A.l.a. OKERCiIEW OF MALE TESTICULAR AND REPRODUCTM TRACT EMBR YOLOGY

I.A. 1 .a.i. DEVELOPMENT OF TESTES

The gonads are derived from three sources: coelomic epithelium, mesenchyme, and

primordial germ cells Noore, 1989bl. They are f h t recognized during the f i f i week of

gestation when a thickened area of coelomic epithelium develops on the media1 aspect of

the mesonephros. Proliferation of these epithelial cells produces a bulge on the mediai side

of each mesonephros called the gonadal ridge. Primary sex cords grow fiom the gonadal

ridges into the underlying mesenchyme. The indifferent gonad now has an outer cortex and

an inner medulla. The cortex will normally differentiate into an ovary while the medulla

----- - - - -- - - - - , -- -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - i - -

sex chromosome constitution, the medulla norrnally differentiates into a testis and the

cortex regresses. Primordial germ cells migrate along the dorsal mesentery of the hindgut to

the gonadal ridges and become incorporated in the primary sex cords [Witschi, 1948;

Peters, 1 9701.

The short a m of the Y chromosome (Yp) carries the sex determining gene, SRY,

which encodes the testis determinhg factor, TDF. TDF is essential for differentiation of the

gonad into a testis and its absence renilts in formation of the gonad into an ovary [Disteche

et al., 1986; Affara et al., 1987; Page et al., 1987; Palmer et al., 1989; Sinclair et al., 1990;

Berkovitz et al., 19911. TDF pennits the prirnary sex çords to differentiate into

seminiferous tubules. The walls of the seminiferous tubules are composed of Sertoli cells,

derived fiom the surface epithelium, and spermatogonia, derived fiom the primordial germ

cells. The Sertoli cells produce Mullerian inhibitory factor (MF) [Josso, 19861. The

seminiferous tubules become separafe- by mesenchyme that gives rise to the interstitial

cells (of Leydig). These cells produce testosterone [Siiten and Wilson, 1 9741.

I.A. l .aii. DEVELOPMENT OF MALE GENITAL DUCTS

Testosterone stimulates development of the Wolffian duct into the male

reproductive tract while MIF suppresses devetopment of the pararnesonephric (Mullerian)

ducts into female genital ducts Moore, l989bI. When the mesonephros degenerates, some

mesonephric tubules near the testis persist and become efferent ductules which open into

the mesonephric duct, forrning the head of the epididymis. The mesonephric duct proper

separates into its ureteral and reproductive divisions at approximately the seventh week of

- - -- -- - - - -. - - - - . - - - - - - - - - - - - - - - - - Y d Y

the metanephric mesodem. The reproductive portion of the mesonephric (Wolfian) duct

forms the body and tail of the epididyrnis. vas deferens, seminal vesicle, and ejaculatory

duct.

I.A. 1 .a.iii. DEVELOPMENT OF MALE EXTERNAL GENITALLA

Masculinization of the indifferent extemal genitalia results fkom androgens

produced by the fetal testes. As the phallus elongates to form a penis, the urogenital folds

fuse with each other dong the ventral surface of the penis to form the penile urethra.

Consequently, the extemal urethral meatus rnoves to the glans penis. The scrotum is

formed by the fusion of the labioscrotal swellings. Feminization of the indifferent external

genitalia occurs in the absence of androgenic stimulation. The final form of the external

genitaiia is established by the twelflh week of gestation Moore, 1989bJ.

I.A.1.b. OVERCIEW OF MALE REPRODUCTIVE PWYSZOLOGY

The testis has two important iùnctions: spermatogenesis in the seminiferous tubules

and secretion of steroid hormones (androgens) by the interstitial Leydig cells. These are

intimately related, as testosterone synthesis is necessary for both spenn production and

development of secondary sexual characteristics. The two functions are oonûolled by the

anterior pituitary through the secretion of gonadotropins. The anterior pituitary itself is

regulated by gonadotropin-releasing hormone (GnRH) fkom the hypothalamus. The

hypothalamic-pituitary-testicular axis consists of a closed-loop feedback control mechanism

for maintaining normal reproductive fiction.

The hypothalamus receives messages from the central nervous system and the testis

to regulate ihe synthesis and secretion of GnRH, which stimulates the synthesis and release

of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [Schally et al.,

19711. These two hormones are synhesized in the anterior pituitary and are secreted

episodically in response to the pulsatile release of GnRH. LH and FSH bind to specific

receptors on the membrane of Leydig cells and Sertoli cells, respectively, to stimulate

cellular metabolism.

The hypothalamic-pitulw-gonadal axis consists of a closed-loop feedback control

mechanism. Testosterone, secreted by the Leydig cells in testes, inhibits LH secretion in

males [Walsh et al., 19731. It is metabolized in peripherai tissue to dihydrotestosterone

(DHT) or estradiol. Secretion of FSH is inhibited by inhibin, a nonsteroidal compound

produced by the Sertoli cells p a n Thiel et al., 19721.

I.A. 1 .b.ii. TKE TESTIS

I.A. 1 .b.ii.a. Leydig Cells

Testosterone is secreted episodically fiom the Leydig cells in response to LH pulses

and has a diurnal pattern, with the peak level in the early morning and the nadir in the

evening [Lipsett, 19741. The biotogic effects of androgens are exerted on target organs that

contain a specific androgen receptor protein in the ce11 cytosol. Testosterone enters target

cells where it may be converted to the more potent androgen, DHT, by Sa-reducbse, and

either testosterone or DHT binds to a receptor protein. This bound receptor complex is

ûanslocated into the nucleus, where it binds to nuclear chromatin and resdts in the

mRNA then exerts its androgen action in the target ce11 [McClure, 19871.

The major functions of androgen in target tissue include regulation of gonadotropin

secretion by the hypothalarnic-pituitary axis, initiation and maintenance of spermatogenesis,

differentiation of the interna1 and extemal male genital system d d n g fetal development,

and promotion of sexual maturation at puberty.

I.A. 1 .b.ii.b. Seminiferous Tubules

Seminiferous tubules contai. germ cells and Sertoli cells. The latter are nondividing

support cells found on the basement membrane of the seminiferous tubules and extend

filamentous cytoplasmic ramifications toward the tubular lumen. Sertoli cells are linked by

tight junctions that divide the seminiferous tubule into a basal and adluminal cornpartment.

These tight junctions dong with the myoid cells of the peritubular contractile ce11 layer,

serve to form the blood-testis barrier wussell, 19801. Sertoli cells are believed to provide

structural and metabolic support for the developing spermatogenic cells. They are also

involved in the phagocytosis of excess cytoplasm cast off by spermatids [Wheater et al.,

19871.

The germ cells are arranged in an orderly manner fiom the basement membrane to

the lumen. Spermatogonia lie d'mtly on the basement membrane, and found progressing

centrally are prirnary spermatocytes, secondary spermatocytes, and spermatids [Clermont,

1963; Heller and Clermont, 19641.

Spematogenesis is the process in which primitive stem cells either divide to renew

themselves or produce daughter cells that will become spermatocytes. The most primitive

undifferentiated spermatogonia are the stem cells. For stem ce11 renewal, the dark type A

spematogonia foltowing mitotic division produce a fiesh stock of type A cells as well as

pale type A spermatogonia. The latter undergo mitotic divisions into preleptotene primary

spermatocytes via type B spermatogonia [Clermont, 19721. The primary spermatocytes

undergo the first maturation division by meiosis, reducing the number of chromosomes

fiom 46 to 23 [Kerr and deKretser, 198 Il. Each prirnary spermatocyte gives nse to two

secondary spermatocytes, and each of these divide into two spermatids. The spermatids

then transfom into spexmatozoa, a process called spermiogenesis. This process includes

nuclear condensation, acrosome formation, loss of cytoplasm, development of a tail, and

arrangement of mitochonciria into the middle piece of the sperm. The entire spermatogenic

process requires approximately 64 days to complete [Clemonf 19721.

I.A. 1 .b.iii. HORMONAL CONTROL OF SPERMATOGENESIS

LH indirectly affects spermatogenesis in that it stimulates endogenous testosterone

production p f i r g a and Sherins, 198 11. Sertoli cells, possessing specific hi&-aninty

FSH receptors, are the target for FSH Neans et al., 1980; Ritzen et al., 19811. Androgen-

binding proteins produced by Sertoli cells carry androgens intracellularly [Ritzen et al.,

1971; Hansson and Djoseland, 19721. The close proxirnity of the Leydig cells to the

seminiferous tubule and elaboration by the Sertoli cells of androgen-binding protein cause a

sperrnatozoa.

I.A. 1 .b.iv. SPERM MATURATION, STORAGE, AND TRANSPORT

While the testis is responsible for sperm production, the epididymis is involved in

the maturation, storage, and transport of spennatozoa. Spermatozoa develop an increased

capacity for motility pedford et al., 1973; Moore et al., 19831 and fertilization [Orgebin-

Cnst, 1969; Bedford et ai., 1973; Hinrischsen and Blaquier, 1980; Moore et al., 1983;

Bedford, 19881 as they migrate through the epididymis.

The epididymis consists of a single convoluted tubule, 3 to 4 metres long, and is

divided into the caput (head), corpus (body), and cauda (tail) epididymis [Jenkins et al.,

19783. Sperrn are transported by a hydrostatic pressure difference, ciliary propulsion, and

peristaltic contraction of myoid cells dong the epididymis [Johnson and Howards, 19761.

The epididymis aiso functions as a storage reservoir for sperm [Johnson and Varner, 19881.

Mature sperm fkom the cauda epididiyrnis enter the vas deferens, which transports

its contents by peristaltic motion into the ejaculatory duct [Bruschini et al., 19771. Sperm

are then propelled to the outside by emission and ejaculation. Secretions fiom the seminal

vesicles and prostate are deposited into the postenor urethra during emission. Peristaisis of

the vas deferens and contraction of the bladder neck are under sympathetic control of the

autonomie nervous system [Bell and McLean, 1 967; Owman and Sjo berg, 1 972; Lipshultz

et al., 19911. During ejaculation, the extemal sphincter relaxes and the semen is propelled

through the urethra by rhythrnic contractions of the perineal and bulbourethral muscles, both

under somatic control.

(20%). The seminai vesicles produce fructose, prostaglandins, and coagulating factors

[Mann and Mann, 19811. Seminal plasma acts as a buffer on the acidic vaginal secretions.

The coagulum formed by the ejacuiated semen liquefies within 20 minutes from the action

of prostatic proteolytic enzymes. More fluid are contributed by the bulboureùiral glands

(Cowper's glands) and urethrai glands (Littre's glands) through the penile urethra. The first

portion of the ejaculate contains most of the spermatozoa and secretions fiom the

bulbourethral glands and the prostate; the remaining portion consists mostly of seminal

vesicle secretions and contains only a few spermatozoa milja et al., 19871.

I.A. 1 . b.v. FERTILIZATION

Fertilization normally occurs in the fallopian tubes after ovulation. During mid-

cycle of the menstnial period in the fernale, cervical mucus becomes more abundant,

thinner, and watery, thus allowing entry of spem into the uterus and also protecting spem

fiom the acidic vaginal environment parnes and Toot, 19861. Spermatozoa must undergo

capacitation within the female reproductive tract in order for fertilization to take place

[Chang, 195 1; Austin, 195 11. Capacitation is followed by the acrosome reaction. The

latter is a progressive fusion between the b e r and outer acrosome membrane, resuiting in

the release of enzymes important for oocyte penetration by the spemi Noore, 1989aJ.

LA.1 .c. CL13VICAL ASPECTS OF W E llVFERTILITY

Pregnancy occurs in the majority of couples within one year of natural unprotected

intercourse. The term infertility applies to those couples who have not been able to

conceive d e r this one year period. Both partners should be evaluated for factors that may

entirely to a male factor, and an additional 20% involve both males and female factors; thus,

about one half of infertile unions is due to a male factor [Mosher, 1985; Sigman and

Howards, 19921.

I.A. 1 .ci . EVALUATION OF THE INFERTILE MALE

The work-up of a man presenting with infertility should consist of a detailed history

and physical examination. The latter must include inspection and palpation of the scrotum

to ensure that the testicles have properly descended into the scrotum and are normal in size

and consistency, and that the epididyrnides and vasa deferentia are present. Abnormal or

suspicious findings fiom the history and physical examination direct subsequent

investigations, such as serum hormonal tests (e-g., TSH, LH, FSH, testosterone, prolactin)

and/or radiological imaging studies (e.g., scrotal ultrasound, transrectal ultrasound).

However, al1 men presenting with infertility should have a semen analysis.

I.A. 1 .c.i.a. Semen analysis

The semen analysis is the cornerstone of the infertility investigation [S ipan and

Howards, 19921. Normal parameters are show in Table 1 [World Health ûrganization,

1 9871. Major abnonnali ties in semen analysis include the following :

asthenospermia (-30% progressively motile sperm)

oligospemiia (QO x 1 o6 sperm 1 mL)

pyospermia (>1 x 106 white blood ce11 1 rnL)

azoospennia (no sperm)

teratospermia (abnormal spem morphology)

I.A. 1 . c i . ETIOLOGIC CLASSIFICATION OF MALE INFERTILITY

The causes of mde infertility may be classified according to results of the semen

analysis. Approximately 10% of men presenting with infertility will have azoospermia,

whereas the remaining 90% will have oligospemia, asthenospermia or teratospermia

[Lipshultz, 19801. Of the patients with azoospermia, standardard laboratory and irnaging

studies can determine whether this abnormality is due to a pre-testicular (hypothalamic-

pituitary pathology, -10% of azoospermie men), testicular (spermatogenic deficiency

including testicular failure, -40%), or pst-testicular (functional or structural obstruction to

spem outflow, -50%). Specific causes of pst-testicular avoospermia include congenital

absence of vas deferens (CAVD), epididymal obstruction, and vasectomy.

I.A. 1 .c.iii. TREATMENT OF MALE INFERTILITY

Obviously, treatment of an individual male patient with infertility will depend on the

underlying cause. Those with a pre-testicda. cause can ofien be treated successfûlly with

exogenous GnRH analogues or gonadotropins mowards, 19951. Advances in rnicrosurgery

and assisted reproductive technologies (ARTS) now make it possible for rnany infertile men

with a testicular or pst-testicular cause to father their own children. Men with previous

vasectomies wishing to reverse their sterile status can undergo microsurgical

vasovasostomy (re-connecting the vas deferens) or, if necessary, vasoepididymostomy

(connecting the vas deferens to the epididyrnis). The latter procedure is also indicated in

cases of acquired (usually due to infection) or idiopathic epididymal obstruction. Men with

CAVU can be ettectively treated with microsurgicai epididymal sperm aspiration

(MESA) and routine in vitro fertilization (NF) or in vifro fertilization-intracytoplasmic

sperm injection (IVF-ICSI) [Temple-Smith et al., 1985; Silber et al., 1988; Palenno et al.,

1992; Silber et al., 1995; Schlegel et al., 1995; Gil-Salom et al., 19951. Patients with a

testicular cause for their infertility such as testicular failure may also be treated with IVF-

ICSI via testis-retrieved haploid sperm [Van Steirteghem et al., 1993; Vanderzwalmen et

al., 1995; Fishel et al., f 995; Fishel and Thornton, 1995; Harari et al., 1995; Silber et al.,

1995; Sherins et al., 1995; Tesarik et al., 19951. However, it must be emphasized that

ARTS should not be initiated until the patient and his partner have received rigorous

genetic counseling on the possibility that the genetic defect responsible for his

reproductive failure may be transrnitted to the offspring [de Kretser, 1995; Ln't Veld et al.,

1995; Mak and Jarvi, 1996; Morris and Gleicher, 19961.

I.A.2. CYSTIC FIBROSIS AND THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE RIEGULATOR

Cystic fibrosis (CF) is the most common fatal autosomal recessive disorder in the

Caucasian population, with an incidence of approxirnately 1 in 2,500 live births and a

carrier fiequency of 1 in 25 persons in populations of Northem European descent. The

classical form of CF is characterized by abnormalities of electrolyte, fluid and

macromolecule secretion of exocrine glands. Clinical hallmarks of CF include chronic

pulmonary obstruction and infections, exocrine pancreatic insufficiency, neonatal

meconium ileus, elevated sweat electrolytes, and male infertility [Welsh et al., 19951.

The gene responsible for CF, called cystic fibrosis transmembrane conductance

regulator (CFTR), was identified and cloned in 1989 [Rommens et al., 1989; Riordan et

- - -

-230 kilobases (kb) of genomic DNA, and produces a mRNA transcnpt of 6.2 kb. The

encoded protein contains 1,480 arnino acids and is a member of the ATP-binding cassette

(ABC) family of membrane proteins. Various studies support that CFTR is a CAMP-

regulated chloide channel found in the apical membrane of secretory epithelial cells [Welsh

et al., 19951.

Hundreds of mutations in the CFTR gene have been reported [Cystic Fibrosis

Genetic Analysis Consortium, 19941. These mutations may be classified as follows: class

1 mutations confer defective protein production; class II mutations involve defective

protein processing; class III mutations involve defective channel regdation; class IV

mutations involve defective channel conduction; and class V mutations lead to decreased

protein synthesis [Zielenski and Tsui, 19951. In general, class 1, II or HI mutations are

expected to have more serious phenotypic consequences than class IV or V mutations.

I.A.3. CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR AND MESONEPHRIC DUCT ANOMALIES

Studies have shown that more than 95% of CF men have abnorrnalities in the

structures derived fiom the Wolfian duct [Kaplan et al., 1968; Valman and France, 1969;

Landiing et al., 1969; Holsclaw et al., 1971 ; Taussig et ai., 19721. The body and tail of

epididymis, vas deferens, seminal vesicles and ejaculatory ducts are atrophic, fibrotic, or

completely absent. As a result, semen analysis typically reveals decreased volume,

complete absence of spermatozoa (azoospermia), increased acidity, decreased fructose

concentration, elevated levels of citric acid and acid phosphatase Kaplan 19681.

However, spermatogenesis is present on testicular histology [Denning, 19681.

Wolffian duct abnormalities but no other manifestations of CF. These conditions may

represent either a reproductive tract specific or a very mild form of CF. At present,

congenital bilateral absence of the vas deferens (CBAVD), congenital unilateral absence

of the vas deferens (CUAVD), and idiopathic epididymal obstruction have al1 been

associated with CFTR gene mutations [Durnur et al., 1990; Anguiano et al., 1992; Mickle

et al., 1993; Osborne et al., 1993; Patrizio et al., 1993; Culard et al., 1994; Oates and

Amos, 1994; Casals et al., 1995; Chillon et al., 1995; Costes et al., 1995; Durieu et al.,

1995; Jarvi et al., 1995; Mercier et al., 1995; Zielenski et al., 1995b; Schlegel et al., 1996;

Donat et al., 19971.

CBAVD is found in 1-2% of men presenting with infertility [Dubin and Amelar,

197 1 ; Greenberg et al., 1978; Jequier et al., 19851. Pnor to the identification of the CFTR

gene, CBAVD was considered to be a distinct clinical and genetic entity with an

autosomal recessive inheritance pattern [McKusick, 19921. Genetic studies found that

5042% of CBAVD men have at least one detectable CFTR gene mutation and that about

15% of these men have two detectable CFTR mutations [Durnw et al., 1990; Anguiano et

al., 1992; Osborne et al., 1993; Paûizio et al., 1993; Culard et al., 1994; Oates and Amos,

1994; Casals et al., 1995; Chillon et al., 1995; Costes et al., 1995; Durieu et al., 1995;

Jarvi et al., 1995; Mercier et al., 1995; Zielenski et al., 1995b; Schlegel et al., 1996;

Donat et al., 19971. CFTR gene mutations have also been reported in men with

congenitai unilateral absence of vas deferens (CUAVD) [Mickle et al., 1993; Schlegel et

al., 1 9961. Of 14 infertile patients with CUAVD tested for 20 CFTR mutations, 6 (43%)

had one mutation, and, in fact, one of these patients had two brothers with CBAVD

CFTR gene mutations was further extended by the observation that 47% of otherwise

healthy men with idiopathic epididymal obstruction have a CFTR gene mutation [Jarvi et

al., 19951. These findings suggest that a broad spectrurn of Wolffian duct abnormalities

(absence of the vas deferens, obstruction of the epididymis, ejaculatory duct cysts,

seminal vesicle cysts, etc.) may be associated with CFTR gene mutations.

Alterations in the CFTR gene identified in men with Wolffian duct abnomalities

can be divided into four groups. The first group consists of mutations that have been

identified in classical CF patients. These "severe" CFTR mutations associate with

pancreatic insufficiency (CF-PI) and are generally class 1-III mutations. Examples of such

mutations reported include AF508, G542X, R553X, R1 l62X, W l282X, N1303K, 1717-

1 G+A, and 2 1 84delA. The second group consists of mutations found in more benign

presentations of CF. These "mild" CFTR mutations are associated with pancreatic

sufficiency (CF-PS) and tend to be class N-V mutations. Such mutations include R117H

and R347P. The third group consists of novel mutations identified exclusively in some

CAVD men; however, as these sequence alterations are extremely rare, it is only

speculated that they contribute to the CAVD phenotype [Anguiano et al., 1992; Culard et

al., 1994; Mercier et al., 19951. The fourth group consists of CFTR gene alterations that

were previously considered as benign sequence variations [Anguiano et al., 1992; Mercier

et al., 1995; Zielenski et al., 1995aI. Such examples include R75Q, G576A, R668C, and

5T. in the majority of patients (-45-50%), only one mutation or sequence alteration has

been identified despite exhaustive screening of al1 exons and immediate flanking intron

sequences in the CFTR gene. For those with mutations identified in both CFTR alleles

It is reasonable to assume that othewise healthy men with CAVD with one or no

detectable CFTR mutation may harbour as yet unidentified CFTR gene mutations or

variants. However, not al1 cases of congenital Wolffian duct anomalies are due to

mutations in the CFTR gene. There is evidence that CUAVD or CBAVD men with

concomitant renal aplasia or ectopia (not typical features of CF) do not result fiom CFTR

gene mutations. Augarten et al. reported that of 47 CBAVD patients, 10 had rend

malformations on ultrasonography [Augarten et al., 19941. Of the 37 patients with

normal urinary tract, 18 (49%) tested positive for CFTR mutations. Interestingly, none of

the 10 individuals with renal malformations had mutant CFTR aileles or abnonnal sweat

tests. in a separate report, 12 of 70 men with CUAVD or CBAVD had renal anomalies

on ultrasound or excretory urography, and al1 12 tested negative for CFTR mutations. Of

58 patients without rend abnormalities, 46 underwent CFTR mutation analysis and 27

(59%) tested positive for mutations [Schlegel et ai., 19961. Therefore, cases with vasal

agenesis and rend malformations probably do not represent a genital form of CF but

rather a distinct clinical entity.

The aforementioned information provides insight into the timing for the effect of

the CFTR mutation on the reproductive tract. The mesonephric duct separates into its

ureteral and reproductive divisions at approximately week seven of gestation. The

ureteral bud induces rend formation as it grows into the metanephric mesoderm, while

the reproductive portion (Wolfian duct) forms the corpus and cauda epididymis, vas

deferens, serninal vesicle, and ejaculatory duct [Moore, 1989bl. It has been proposed that

in CUAVD or CBAVD not caused by a defect in the CFTR gene, anomalous changes to

-

and genital systems are afFected [Augarten et ai., 19941. In CUAVD or CBAVD patients

with CFTR mutations. changes to the Wolffian duct are believed to occur after the urinary

system has separated fiom the reproductive system; therefore. no renal malformation

resuits. n e pathogenesis probably involves intrauterine obstruction of the Wolffian duct

with inspissated secretions which results from CFTR chloride channel dysfunction, and

gives rise to a dehydrated intraluminal fluid content with secondary degeneration or

obliteration of the Wolffian duct [Taussig et al., 19721. However, a primary

morphogenetic defect in development of the Wolfian duct cannot be excluded [Olson

and Weaver, 19691.

Electrophysiological experiments on cultured hurnan epididymal cells using the

patch-clamp technique demonstrated the presence of a low-conductance chloride channel

that was activated by addition of CAMP agonists to these cells pollard et al., 19911. This

channel most likely represents CFTR. Furthemore, expression studies in human fetal

and adult reproductive tract by in situ hybndization showed that CFTR is expressed in the

ductal epithelium by week 18 of gestation and th is expression is maintained throughout

pst-natal life [Trezise et al., 1993; Tizzano et al., 19931. CFTR expression is high in the

head of the epididymis, low in the body and tail of the epididymis, low in the vas

deferens, and undetectable in the seminal vesicles [Tizzano et al., 19941. The decreased

expression of CFTR in Wolffian denvatives suggests that these structures may be more

sensitive to CFTR dysfunction than other organs.

SPLICING OF CFTR EXON 9

One of the first forms of alternative splicing discovered in CFTR was the deletion

of exon 9 (exon 9-) [Strong et al., 1991 ; Chu et al., 19911. Exon 9 encodes the initial

21% of the first nucleotide binding fold (NBFI) of CFTR, including motif A of the

Walker ATP binding consensus sequence [Walker et al., 19823. Based on quantitative

RT-PCR experirnents performed on respiratory epithelia, the amount of exon 9-

transcripts was found to Vary between different individuals [Bremer et al., 1992; Chu et

al., 1991; Chu et al., 19921. The variability in exon 9 splicing efficiency can be

explained, for the most part, by a variation in the polypyrimidine tract of the splice

acceptor site in intron 8 of CFTR (IVS8-T tract) [Chu et al., 19931. Sequencing of the

splice acceptor site preceding exon 9 demonstrated that three variations of uninterrupted

pyrimidines occur in the population: 5,7, and 9 thymidines (5T, 7T, and 9T) [Chu et al.,

19911. It was also observed that a positive correlation existed between the length of

IVS8-T tract and the proportion of branchial epithelial CFTR transcripts with exon 9

intact (exon 9+) (Fig. 1) [Chu et al., 19931.

The abundance of exon 9- CFTR eanscripts in respiratory epithelial cells lead two

groups of investigators to determine whether it encoded a functional isoform of CFTR.

Transduction of CFPAC (pancreatic adenocarcinorna ce11 line from a CF patient) or HeLa

cells with the CFTR cDNA lacking exon 9 failed to confer CAMP-regulated chloride

conductance [Delaney et al., 1993; Strong et al., 19931. Protein analysis of transduced

cells detected mutant CFTR that was immature and incompletely glycosylated in both

studies, suggesting that exon 9- CFTR mRNA transcripts are û-anslated but the protein

- - - -

protein probably undergoes degradation within the endoplasmic reticulum. Expression of

the CFTR missing exon 9 encoded amino acids in Xenopus oocytes incubated at room

temperature also did not produce a CAMP-regulated chloride conductance. This finding

is important as other CFTR mutants that are improperly processed at 37OC appear to fold

properly and regain function at lower temperatures [Drumm et al., 1991; Denning et al.,

19921. Although some have argued that the protein made by the exon 9- transcript may

have physiological function [Bremer et al., 19921, the lack of conservation of exon 9-

transcripts in mice suggests otherwise [Delaney et al., 19931. Thus, CFTR missing the

region of NBFl encoded by exon 9 does not function as a chloride channel and likely

does not have any intracellular function.

I.A.5. THE 5T VARIANT IN INTRON 8 OF THE CFTR GENE AND CONGENITAL ABSENCE OF VAS DEFERENS

Studies have shown that the fiequency of the 5T variant was significantly higher

in men with CBAVD (46%), CUAVD (25%), or epididymal obstruction (29%) than the

general population (5%) [Chillon et al., 1995; Costes et al., 1995; Jarvi et al., 1995;

Zielenski et al., 1995bl. Indeed, the 5T variant is the single rnost fkequent CFTR gene

sequence alteration found in CBAVD men (Table 2). The presumed role of the 5T allele

in the pathogenesis of CBAVD is supported by two separate studies showing that the

frequency of the 5T allele is lower in fathers (2.1 % and 1.9%) than in mothers (5.2% and

4.7%) of CF patients [Chillon et al., 1995; Zielenski et al., 1995bl. In fact, Chillon et al.

reported four fathers who had one CFTR gene with the 5T ailele and the other with a

severe CFTR mutation (G542X, Nl3O3KY 18 12-1 G+A, or 936delTA) [Chillon et al.,

and are phenotypically normal. Three hypotheses could explain the strong but incomplete

association between the 5T allele and CBAVD. First, there could be a non-random

association between 5T and CBAVD, with the allele segregating with but not being

causal for the CBAVD phenotype; however, this hypothesis is probably untrue as the

analysis of several intragenic DNA markers (Le., within the CFTR gene) in the four

fathers and in patients with CBAVD showed that several haplotypes are associated with

the 5T allele [Chillon et al., 19951. Second, the 5T variant may have a partially causal

role, together with other CFTR gene mutations. The presence of another mutation in the

same CFTR gene as the 5T allele is unlikely since al1 coding regions and their

immediately flanking intron sequences have been screened [Chillon et ai., 1995; Costes et

al., 1995; Zielenski et al., 1995b3. However, this does not d e out mutations within the

promoter region or introns of the CFTR gene. Third, the ST variant could have a causal

role in CBAVD, with other factors (genetic andor environmental) accounting for these

exceptional cases without CBAVD (Le., the four fathers with the CFTR rnutatiod5T

genotype). The association of the 5T allele with lower levels of normal, full-length

CFTR mRNA supports the hypothesis that this variant generally causes CBAVD,

especially when a CFTR mutation is present in the other copy of the gene. Nevertheless,

the association between the 5T allele and CBAVD is not absolute. Taking into account

the predicted frequency (1 in 222 men) and the reported iiequency (-1 in 1,000 men) of

CBAVD, it has been estirnated that the penetrance of the 5T allele is -0.6 wielenski et

al., 1995bl. While confirmation of this estirnate awaits M e r studies involving larger

penetrance.

I.B. HYPOTHESIS

An intriguing question pertains to the mechanism by which dterations in CFTR

expression results in reproductive tract abnormalities in the absence of pathologie

changes in other CF-associated organs (e.g., lung, nasal sinus, pancreas, liver). It is

possible that the reproductive tract is more sensitive to CFTR dysfunction than the other

tissues. Alternatively, the level of functional CFTR may vary between different tissues,

with the lowest expression in the Wolfian structures. Since the 5T variant of the NS8-T

tract of the CFTR gene is associated with inefficient splicing of exon 9 and has been

identified in a signifiant proportion of infertile men with isolated Wolffian duct

anomalies, it is proposed that the splicing efficiency of CFTR exon 9 is lower in the

reproductive tract than the other CF-associated organs.

I.C. PURPOSE

The purpose of my work was to compare the proportion of exon 9+ CFTR mRNA

transcripts within nasal epithelia and vas deferens fiom the sarne individual, and to

examine the relationship between the IVS8-T tract and the level of exon 9+ CFTR rnRNA

in these tissues.

1I.A. PATIENTS AND SAMPLES

Twenty-four healthy men with a previous vasectomy presenting to a male

infertility clinic for sterilization reversai were recniited for the study (mean

agefSD4Of3 years). This study was performed with the approval of the Human

Subjects Review Cornmittee of the University of Toronto, and informed consent was

obtained fiom each subject. None of the patients had a personai or family history of

pulmonary or gastrointestinai manifestations suggestive of CF. Each patient underwent

scrotal surgery (i.e., either vasovasostomy (re-comecting the vas deferens) or

vasoepididymostomy (connecting the vas deferens to the epididymis)). Intraoperatively,

while under general anesthesia, in each case the following were obtained: peripherai

venous blood by venipunchire for analysis of the NS8-T tract genotype, nasal epithelia

by a sterile swab and a small piece of vas deferens (-5- 10 mm, which would ordinarily be

discarded) by excision for quantitative RT-PCR analysis. Nasal samples were placed into

a 2 ml microfuge tube containîng 500 pl of phosphate buffered saline and vasal sarnples

were placed into a 2 ml microfuge tube, snap-fiozen in liquid nitrogen, and stored at -

70°C if not analyzed immediately.

1I.B. ALLELE SPECIFIC OLIGONUCLEOTIDE ANALYSIS OF IVSS-T TRACT

Genomic DNA was isolated fiom peripheral blood lymphocytes according to

standard protocols [Miller et al., 19881. Exon 9 including the WS8-T tract was amplified

by the polyrnerase chah reaction (PCR) mullis et al., 19861 with primers 9i-5 (5 ' -

TAATGGATC ATGGGCC ATGT -3 ' ) and 9i-3 (5 ' -AC AGTG'TTGAATGTGGTGC A-3 ' ),

follows: 94OC for 2 min; denaturation at 94OC for 20 S. annealing starting at 60°C then

auto-down OS°C/s over 20 s, and extension at 72OC for 30 s, for 20 cycles; followed by

denaturation at 94OC for 20 s, annealing at 50°C for 20 s, and extension at 72OC for 30 s,

for 15 cycles; followed by one final cycle of denaturation at 94OC for 20 s, annealing at

50°C for 20 s, and extension at 72OC for 7 min. The reaction mixture contained 5 pl of

PCR buffer (10 mM Tris-HC1 (pH 8.3), 50 rnM KCl, 1.75 mM MgC12, 0.001% gelatin), 5

pl of dNTP's (2 m M each of deoxyadenine triphosphate, deoxycytidine triphosphate,

deoxyguanosine triphosphate, and deoxyrhymidine triphosphate; Pharmacia); 50 ng of

each primer; and 0.5 unit of Taq DNA polymerase (Perkin Elmer/Cetus) in a final volume

of 50 pl, containing 50-100 ng of genomic DNA. Al1 PCR amplifications were

performed with the GeneAmp PCR System 9600 (Perkin ElmerlCetus). Ten pl of PCR

arnplified DNA was denatured and vacuum-blotted to nylon membrane (Hybond-N+;

Amersharn). The following allele specific oligonucleotides were used: ST (5 ' -

GTGTGmAACAGG-3') , 7T (5'-GTGTGTTTTTTTAACAG-3'), and 9T (5'-

TOTGmAAC AG-3'). Each oligonucleotide was labeled with 3 2 ~ by terminal

transferase (Pharmacia), and hybridization was performed ovemight at 42OC and the

washings were done once with 3xSSC/O.l%SDS at room temperature for 20 min, and

twice with 0.2xSSC at 36OC for 20 min. Amplified DNA fiom three individuals

previously s h o w to have the 5T/7T, 7T/7T, or 9T/9T genotype were used to control for

oligonucleotide specificity [Jarvi et al., 19951.

For reconstruction studies, a segment of exon 9+ cDNA was obtained fiom a

plasmid containing full-length CFTR cDNA [Rommens et al., 19911 following two

rounds of nested PCR. First round PCR was performed with plasmid wild-type CFTR

cDNA, 5' primer X5-5 (5'-GCTGTCAAGCCGTGTTCTAG-3'' in exon 5) and 3' primer

13i-3sA (5'-TGGTCGAAAGAATCACATCC-3', in exon 13) for 35 cycles (94OC, 20 s;

60°C, 20 s; 72OC, 30 s) (Fig. 3A). The reaction mixture contained 5 pl of PCR buffer (10

rnM Tris-HCl (pH 8.3), 50 mM KCl, 1.75 m M MgCI2, 0.001% gelatin), 5 pl of dNTP's

(2 rnM of each of the four nucleotides triphosphate; Pharmacia); 200 ng of each primer;

and 0.5 unit of Taq DNA polymerase (Perkin ElmerKetus) in a final volume of 50 pl,

containing 400 ng of plasmid wild-type CFTR cDNA. Nested PCR was performed on 115

of the reaction product fiom the first round under identical PCR conditions, except that 5'

primer 7i-5s (5'-TTCAATAGCTCAGCCTTC-3', in exon 7) and 3' primer X12-3 (5'-

GTTAAAACATCTAGGTATCC-3', in exon 12) were used. The PCR products were

size fiactionated on 1% agarose gel in 1 X TAE (40 mM Tris-acetate; 1 mM EDTA, pH

8.0). The exon 9+ cDNA fhgments were extracted fiom the agarose gel and purified

according to the manufacturer's protocol (QIAquick Gel Extraction KitlQLAGEN). Exon

9- cDNA fragments were isolated fkom commercially-obtained hurnan lung total RNA

(Clontech) after conversion to cDNA and amplification by two rounds of nested PCR as

described above, except the exon 9- hgments were extracted fiom the agarose gel and

purified (Fig. 3B).

POLYMERASE CHAIN REACTION

Nasal epithelial cells were collected by centrifugation and lysed in 800 pl of

TRIzot Reagent (Gibco/BRL). Vas deferens sarnples were homogenized with a rotor-

stator homogenizer (PRO 200 Handheld Homogenizer, PRO Scientific Inc.) in 800 pl of

TNzol Reagent. Total RNA was extracted fiom the lysates according to the

manufacturer's instructions (Gibco/BRL). First strand cDNA was synthesized fiom total

RNA using Superscript II RNase H- reverse transcriptase (RT) and random

hexanucleotide pnmers. The reaction (21 pl) contained up to 1 1 pl of total RNA, 2 pl

lOxPCR buffer (100 mM Tns-HCl (pH 8.4), 500 rnM K I ) , 2 pl MgCl2 (25mM), 1 pl 10

m M dNTP mix, 2 pl DTT (0.1 M), 1 pl random hexamers (5Ong/pL), 1 pl Superscnpt II

RT (200 unitslpl), and 1 pl E. coli RNase H (2 unitdpl), with al1 components obtained

comrnercially fiom Gibco/BRL.

To ensure that total RNA was successfidly extracted and synthesized into cDNA,

rnRNA of the human housekeeping gene glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) [Tokunaga et al., 19871 fiom each sample was amplified using the PCR

conditions described below. Primers used in the reaction were S'primer GAPDHSB (5'-

GGTCGGAGTCAACGGATTTGGTCG-3') and 3' primer GAPDH3B (5' -

CCTCCG ACGCCTGCTTCACCAC-3 '). The ampli fied products (78 8 bp) were size

fractionated by agarose gel (1.5%) electrophoresis, transferred to Hybond N+ nylon

membrane (Amersham) by the rnethod of Southem [Southem, 19751 and evaluated with a

32~-labeled human GAPDH cDNA probe (GAPDH 5A; 5'-

CCAATATGATTCCACCCATG-3') interna1 to the amplified region. The resultant

3 5 SoftwareProtein Databases Inc).

AAer being adjusted with GAPDH mRNA transcnpt level for each individual

sample, transcripts of the CFTR gene were amplified by PCR. The oligonucleotide

primers included 5 ' primer X8-5 (5 '-ACGACTACAGAAGTAGTGATGGAG-3 ' , in

exon 8) and 3' primer C 16D (5'-GTT'GGCATGCTITGATGACGCTTC-3', in exon 10)

(Fig. SA), and amplification was performed as follows: 94OC for 2 min; denahiration at

94OC for 20 s, annealing at 5S°C for 20 s, and extension at 72OC for 30 s, for 30 cycles;

followed by one final cycle of denaturation at 94OC for 20 s, mealing at 50°C for 20 s,

and extension at 72OC for 7 min. The PCR products were blotted to nylon membrane and

hybridized with a CFTR cDNA oligonucleotide probe derived fiom exon 10, which

would anneal to both exon 9+ and exon 9- fragments (C16B; 5'-

GT'ITTCCTGGATTATGCCTGGCAC-3') (Fig. 5B). The expected size of the exon 9+

and exon 9- products were 4 16 bp and 233 bp, respectively. To ensure that the 4 1 6 bp

and 233 bp fragments respectively represented exon 9+ and exon 9-, two oligonucleotide

probes were designed: 9i-5s (5'-ACAGGGATITGGGGAATTATTTG-3'), a sequence

within exon 9, and ex8/10 (5'-TGGGAGGAGACTTCACTT-3'), a sequence which spans

the 3' end of exon 8 and the 5' end of exon 10 (Fig. SB).

The absence of contaminants in RT-PCR assays was regularly assessed by

controls that did not contain any cDNA template, starting RNA, or RT enzyme.

Nasal epithelial and vas deferens total RNA were extracted, converted to cDNA,

amplified by PCR with CFTR primers, and subjected to Southem analysis using a '*P-

labeled exon 10 probe (C 16B) as described in Section H.D. The resulting autoradiograph

was analyzed by scanning densitometry, and the proportion of exon 9+ transcripts, as a

percentage of total CFTR transcripts, was denved by using the total densitometric units of

both transcripts as 100%.

As standards for quantification of exon 9+ and exon 9- mRNA species, the

isolated exon 9+ and exon 9- cDNA fragments (see Section II.C.) were serially mixed in

varying, known quantities and subjected to the same PCR conditions as for the nasal and

vas deferens samples as outlined in Section U.D. Quantification of exon 9+ and exon 9-

was then determined as described above. The results were plotted and graphed. Al1

experimental proportions of exon 9+ and exon 9- CFTR transcripts were accordingly

adjusted, based on this graph.

1I.G. ANALYSIS OF DATA

Differences in IVS8-T allele frequencies between study subjects and the general

population were compared by the X2 statistic or Fisher's exact test. The mean proportions

of CFTR exon 9+ mRNA transcripts associated with the different WS8-T genotypes were

compared uing analysis of variance (ANOVA). Post-hoc pairwise comparisons between

the different means were evaluated by the Bonferroni test. The correlation between the

proportion of exon 9+ transcripts and length of the IVS8-T tract was analyzed by the

Spearman rank correlation. Intra-individual nasal and vas deferens levels of exon 9+

cornparisons and those less than 0.05 were considered to indicate statistical significance.

Al1 statisticai analyses were performed using SPSS for Windows 1994.

1II.A. IVSS-T TRACT GENOTYPE

Leukocyte genomic DNA was isolated and amplified by PCR using CFTR

primers which encompass the IVS8-T tract and exon 9 region (Fig. 2A). Subsequent

ethidium bromide staining and gel electrophoresis confirmed successfùl amplification

(Fig. 2B). Slot blot analysis using allele specific oligonucleotides for the 5T, 7T, and 9T

variants revealed that, of the 24 patients, 8 were 7Tl9T heterozygotes, 14 were 7Tl7T

homozygotes, and the remaining 2 were 5Tl7T heterozygotes (Fig. 2C). As shown in

Table 3, the fiequencies of the 9T, 7T, and 5T alleles were not significantly different

between individuals in the present study and the general population [Kiesewetter et al.,

1993; Cuppens et al., 1994; Dork et al., 1994; Chillon et al., 19951 (9T: 16.7% vs.

1 1.5%, p=0.28; 7T: 79.2% vs. 83.3%, e . 4 6 ; 5T: 4.2% vs. 5.2%, p=0.75).

1II.B. CONVERSION OF EXPERIMENTAL TO ACTUAL PROPORTIONS OF EXON 9+ CFTR mRNA

To establish accurate measurement of the proportion of exon 9+ CFTR

transcnpts, it was fmt necessary to establish a standard c u v e using known concentrations

of isolated fiagments of exon 9+ and exon 9- transcripts. A plasmid containing Ml-

length CFTR cDNA served as the source of exon 9+ transcnpts pomrnens et al., 19911.

M e r two rounds of nested PCR using primers as shown in Fig. 3A, the exon 9+

fragments were extracted fiom agarose gel and purified. Exon 9- CFTR trmscnpts were

similarly obtained, except iung total RNA served as the source (Fig. 3B). Analysis by

electrophoresis and ethidium bromide staining revealed that a greater concentration of

. -

was then serially diluted and mixed with a constant quantity of the latter, and analyzed by

Southern transfer and scanning densitometry. This demonstrated that the concentration of

the isolated exon 9+ species was higher than that of exon 9- (Fig. 3D). Therefore, the

former was diluted to obtain equivalent concentrations of exon 9+ and exon 9-

transcripts. We then verified our calibration and the purity of the isolated exon 9+ and

exon 9- fragments by subjecting them to Southern analysis and hybridization with both

specific and cornmon radiolabeled oligonucleotide probes (Fig. 3E). Hybridization with

the probe comrnon to both transcripts (C16B) and subsequent densitomeûic anaiysis

confmed that we had accwately obtained equivaient concentrations of the exon 9+ and

exon 9- fiagments (Fig. 3F). Furthemore, hybridization with probes unique for exon 9+

or exon 9- rnRNA, validated the purity of the exon 9+ and exon 9- isolates, as the exon

9+ specific probe (9i-5s) and the exon 9- specific probe (ex8/10) annealed only to the

exon 9+ and exon 9- cDNA hgrnents, respectively (Fig. 3F).

Next, the exon 9+ and exon 9- fiagments were serially mixed in known

proportions and subjected to the identical PCR conditions and quantification protocol as

for the nasal and vas deferens specirnens (Fig. 3G-1). The experimental proportions of

exon 9+ and exon 9- transcripts were derived from densitometric scanning, taking the

surn of densitometric units for the exon 9+ and exon 9- bands as 100%. The

corresponding actual and experirnental proportions of exon 9+ transcripts were then

recorded graphically (Fig. 35). Consequently, al1 experhental proportions of exon 9+

transcripts from our tissue samples were adjusted according to this graph.

Total RNA was extracted fiom nasal and vas deferens specimens, converted to

cDNA, arnplified initially with primers for the transcript of the ubiquitously expressed

GAPDH gene, and analyzed by Southem hybridization. Quantification of the

autoradiographic bands by scanning densitometry showed differences in the yield of RNA

extraction and cDNA synthesis (Fig. 4). This information was then employed to estimate

the volume of cDNA to be used for PCR amplification with CFTR primers. For

example, Fig. 4 illustrates that cDNA obtained fiom the vas of patient 5 was -3.4 times

less than that of patient 1, hence 3.4 times the amount of cDNA fiom patient 5 was used

for the CFTR transcript analysis. These steps minimized inter-sample variability in RNA

extraction and cDNA synthesis, thus allowing cornparison of the proportion of exon 9+

CFTR transcripts within and between subjects.

Evaluation of CFTR mRNA transcripts fiom nasal epithelial and vas deferens

cells in the region encompassing exons 8 to 10 d e r conversion to cDNA and PCR

amplification revealed two different kgments. The difference in size between these two

bands (416 bp and 233 bp) corresponded to the size of exon 9 (183 bp) (Fig. 5A).

Southem analysis with an exon 10 specific probe (C16B) showed that both fragments

contained exon 10 sequences (Fig. 33; Fig. SC, lane 1). However, Southern analysis with

the exon 9+ specific probe (9i-5s) annealeci to the larger fiagrnent only (Fig. 5B; Fig. SC,

lane 2), while the exon 9- specific probe (ex8/10) detected only the smaller transcript

(Fig. 5B; Fig. SC, lane 3). These results confirm that the larger transcript represented the

normal, full-length transcript with exon 9 intact, and that the smaller transcript

represented the in-fi-ame exon 9 deleted transcript.

1ll.U. VUAN'I'Ik'1LA171VN OF EXVfl Y+ CFI'K mKRA

Al1 proportions of exon 9+ transcripts reported hereafter refer to the corrected,

actual proportions, based on Fig. 35. The proportion of exon 9+ nasal transcripts were

93&2%, 82t3%, and 74&1% (memSD) for the 7T/9T, 7T/7T, and 5T/7T genotypes,

respectively, while in the vas deferens sarnples, the proportion of exon 9+ transcripts

were 88&3%, 76k3%, and 64k4% for the 7T/9T, 7T/7T, and 5W7T groups, respectively

(Fig. 6 & 7; Table 4).

1XI.E. RELATIONSHIP BETWEEN LEVEL OF EXON 9+ CFTR mRNA AND THE IVSS-T TRACT

For both the nasal and vas deferens samples, significant differences were noted in

the mean exon 9+ levels for the different IVS8-T genotypes (p<0.0001, ANOVA; p<O.O5,

Bonferroni). In addition, a strong positive correlation was found between the length of

the IVS8-T tract and the proportion of exon 9+ &anscripts in nasal epithelia ( ~ 0 . 8 8 ,

p<0.001, Spearman) and in vas deferens ( ~ 0 . 8 7 , p<0.001, Spearman) (Fig. 7).

1II.F. COMPARISON OF THE PROPORTION OF EXON 9+ CFTR mRNA IN NASAL EPITHELIUM TO VAS DEFERENS

The mean levels of nasal and vasal exon 9+ transcripts were 85I7% and 79I8%

(meankSD), respectively. When evaluating the proportions of exon 9+ transcripts for

these two different CFTR expressing tissues fiom the same subject as paired samples, a

significantly higher proportion of exon 9+ transct-ipts was found within the nasd

epitheliurn compared to the vas deferens (p<0.001, paired t-test) (Fig. 8).

1V.A. SPLICING EFFICIENCY OF CFTR EXON 9 IS RELATED TO THE IVSS- T TRACT LENGTH

In the present study, we found a relationship between the WS8-T genotype and

the proportion of normal, full-length CFTR mRNA ûmscripts (exon 9+) in nasal

epithelia and vas deferens. It appears that the longer the WS8-T tract, the greater the

proportion of normal CFTR transcripts in nasal epithelia and vas deferens cells (Fig. 7;

Table 4). This fmding is consistent with that reported by Chu et al., who also

demonstrated a positive correlation between the proportion of exon 9+ transcripts in

branchial epitheliai cells and the length of the NS8-T tract [Chu et al., 19931. It is likely

that this relationship holds true for other CFTR-expressing tissues as well.

N.B. HlGHER PROPORTION OF NORMAL, CFTR mRNA IN NASAL EPITHELIUM THAN VAS DEFERlENS

Our study aisu showed that there is a greater proportion of the normal, Ml-length

CFTR message in nasal epithelia than in vas deferens fiom the same subject, regardless of

the NS8-T genotype @<0.001, paired t-test) (Fig. 8). In other words, the precise excision

of intron 8 with in-£&ne joining of exon 8 and exon 9 of CFTR *A may be less

efficient in epithelia of the reproductive tract compared to those of the respiratory tract.

Two recent reports corroborate our findings. Teng et al. found that, for the same NS8-T

tract genotype, the proportion of exon 9+ transcripts was lower in a series of vas deferens

samples obtained fiom vasectornized men than in a series of nasal biopsies obtained fiom

different men and women with chronic nasal obstruction or sinusitis [Teng et al., 19971.

Rave-Harel et al. documented that three men with CBAVD had an increased level of exon

19971. Taken together, these and our observations support the hypothesis that splicing

efficiency varies between the different tissues affected in CF.

W.C. THE CFTR WSS-T TRACT MODIFIES PHENOTYPE BY VARIABLE SPLICING OF EXON 9 IN DIFFERENT TISSUES

The discovery of differential splicing effrciency between the various tissues which

express CFTR provides important insights into the relationship between tevels of normal

CFTR and phenotypic variation. For instance, the R117H mutation is associated with

pancreatic-sufficient CF (CF-PS) mst id i s et al., 19921. CF-PS patients with this

mutation have pulmonary dysfunction but do not have pancreatic exocrine insufficiency

and their sweat chloride measurements are only modestly elevated [Harnosh et al., 19941.

Not surprisingly, the rnild R117H mutation has been identified in otherwise healthy males

with CBAVD. However, M e r genetic analysis uncovered that individuals

heterozygous for the R117H mutation on a 5T background (i.e., R117H and 5T on the

same chromosome) and a "severe" CFTR mutation (e.g., AF508, G55 1 D) developed lung

disease characteristic of CF, whereas the RI 17H mutation found in CBAVD men is

associated exclusively with the more efficient splice acceptor 7T (Le., R117H and 7T on

the same chromosome) [Kiesewetter et al., 19931. It is also important to note that the

R117H mutation gives rise to a partially functional CFTR protein [Sheppard et al., 19931.

Therefore, the R117W5T allele results in a low enough level of partially functioning

CFTR in the lung and an even lower level in the reproductive tract such that both organs

are affected. In contrast, the R117W7T allele, although producing a sufficient level of

partially functional CFTR in the lung to prevent disease, the lower level in the genital

results strongly suggest that the specific IVS8-T tract background on which a CFTR

mutation resides can modulate disease severity in a tissue-specific manner.

1V.D. PROPORTION OF NORMAL CFTR mRNA PRODUCED BY THE IVSS-T ALLELES

The frequencies of the 9T, 7T, and 5T alleles of the IVS8-T tract of the CFTR

gene in our study sample are similar to those in the general population wesewetter et ai.,

1993; Cuppens et al., 1994; Dork et al., 1994; Chillon et al., 19951 (Table 3). This

finding is not unexpected as both groups consist of normal, healthy subjects. The fact

that our study lacked subjects with the 9T/9T, 5T/9T, or 5TET genotype is likely a

consequence of the relatively small sarnple size. Despite the absence of these groups,

based on our subjects with the 7T/9T, 7Tl7T and 5T/7T genotypes (Table 4) and the

assumption that each of the two CFTR alleles contributes equally to the total arnount of

CFTR transcripts, it is inferred that the 7T ailele produces -41% exon 9+ transcripts (ie.,

-41% of the total arnount of CFTR transcripts is exon Pt) in nasal epithelium and -38%

exon 9+ transcripts in vasal epithelium. It follows, then, that the 5T allele produces

-32% and -26.5%, and the 9T ailele produces -49.5% and 4 9 % , exon 9+ CFTR mRNA

in nasal epithelial and vas deferens cells, respectively (Table 5). Therefore, although the

present study does not consist of any individuals with the 9T/9T, 5T/9T, or 5T/5T

genotypes, it c m be deduced that they would have 99% and 98%, 82% and 76%, and 64%

and 53%, exon 9+ CFTR transcripts in nasal epithelia and vas deferens, respectively

(Table 6).

phenotype and the amount of normal CFTR message (Fig. 9). A phenotypically normal,

male CF carrier typically has the 7T variant on one chromosome and a severe CFTR gene

mutation (e.g., AF508) on the other. The latter will result in absent or non-functional

CFTR protein while the 7T variant may give rise to -41% normal CFTR in respiratory

tract and -38% normal CFTR in reproductive tract, enough to sustain a normal phenotype

in these tissues. On the other hand, a typical CBAVD patient may harbor the 5T variant

on one chromosome and a severe CFTR mutation on the other. He may produce -32%

normal CFTR in the lung, which is adequate to sustain normal pulmonary f ict ion, but

-26% in the reproductive tract, an insufficient level to confer a normal genital duct

phenotype. However, the occurrence of fertile males with the severe CFTR mutation6T

genotype, such as fathers of some CF patients [Chillon et al., 19951, suggests that other

genetic factors (e.g., expression of dternative chloride channels) andfor environmental

influences can ameliorate the unfavorable eflects of certain CFTR gene sequence

alterations. Alternatively, since a range of exon 9+ mRNA level does exist for the same

ïVS8-T genotype (Fig. 7; Table 4), these heaithy, fertile men with a severe CFTR

mutation and the 5T allele may produce a level of normal CFTR fkom the 5T

chromosome that exceeds a minimal essential threshold. Furthermore, CBAVD men with

the severe CFTR mutatiod5T genotype may harbor additional mutations not detectable

by our curent DNA mutation screening methods (e.g., mutations within the promoter

region or introns of the CFTR gene).

Various studies have reported different mean values for the proportion of exon 9+

transcripts produced fiom the various IVSS-T tract genotypes. For example, with respect

ro me 1 i I I i genorype, u u er ai. reporcea a mean proporcion or exon Y+ nasal epitheliai

transcripts to be 75% [Chu et al., 19931 while Teng et al. docurnented 86% [Teng et al.,

19971. Although these estimates are not significantly different fiom ours (82%), the

slight discrepancy may be partly explained by the different methods used in the

quantitative RT-PCR analysis, such as the nurnber of cycles and nested rounds of PCR

employed, which pairs of primers were used in the PCR reaction, whether oligo-

deoxythymidine or random primers were utilized in the first strand cDNA synthesis, etc.

in addition, these studies employed a differential RT-PCR which may lead to preferential

amplification of the smaller exon 9- cDNA [Walsh et ai., 19923. Recently, Rave-Harel et

al. designed nondifferential RT-PCR reactions in which both exon 9+ and exon 9- cDNA

products were of the sarne size [Rave-Harel et al., 19971. However, this necessitated the

use of different primers and oligonucleotide probes. The former may result in different

amplification efficiencies while the latter will require deprobing and rehybridization

procedures which may lead to inadequate stripping of the previous probe andor some

loss of membrane-bound PCR products. In our approach, we minimize the number of

PCR cycles and introduce a standardization curve which should more accurately reflect

the actual proportion of exon 9+ transcripts (Fig. 3). The methodology involved is simple

and may be applied broadly to other similar quantitative RT-PCR analyses.

N.E. MOLECULAR MECNANISM OF DIFFERENTIAL SPLICING EFFICIENCY OF CFTR EXON 9

Several consensus sequences are found within introns of higher organisms that are

important for the efficient splicing of nuclear pre-mRNA. These include the s'-GU splice

donor, 3'-AG splice acceptor, branch-point A at -20 to 50 bases fiom the 3' splice site,

consensus has eleven consecutive nucleotides consisting of thymidine andfor cytosine

[Krainer and Maniatis. 19881. Extensive polypyrimidine tracts can make these sites more

cornpetitive as splice acceptor sites [Helfman and Ricci, 1989; Smith and Nadal-Ginard,

19891, while deletions in the polypyrimidine tract has been s h o w to inhibit the

S'cleavage reaction [Frendeway and Keller, 1985; Reed and Maniatis, 1986; Ruskin and

Green, 19851, binding of the splicing factors U2A.F and U2snRNP [Ruskin et al, 1 9881,

and assembly of the splicing complex [Frendeway and Keller, 19851. Furthemore, the

observation that identical pre-mRNA transcripts are processed into alternatively spliced

forms in a cell-type specific manner strongly suggests that the splicing environment of

different cells varies for these transcripts [Breibart and Nadal-Ginard, 1987; Wieczorek et

al., 19881. This tissue-specific difference in the splicing environment may be due to the

presence of specific alternative splicing factors or variations in the activities or levels of

constitutive splicing factors. Thus, the short polypyrimidine tract in CFTR intron 8,

especially with ST, likely makes it underutilized as a splice acceptor site, resulting in

reduced efficiency of exon 9 splicing. This effect is presumably more pronounced in the

reproductive tract than in the other CF-associated organs.

1V.F. StJMMAFtY

In sumrnary, o u study confums the hypothesis that splicing efficiency of CFTR

pre-mRNA varies between the different organs affected in CF. Specifically, depending

on the IVS8-T tract background, the splicing efficiency of CFTR exon 9 may be poor in

the genital tract but adequate in other tissues, thus explaining some cases of the CAVD or

- A . - . - . . . . - - - - - - - - - .

However, the existence of fertile males with the 5T genotype [Chillon et al., 19951 imply

that other genetic factors (e.g., expression of alternative chloride channels) and

environmental influences contribute to the overall phenotype. Studies such as ours may

have important future implications for CF gene therapy, which will involve delivery of

the appropriate level of normal CFTR into affected cells. Moreover, the study of the

molecular pathogenesis underlying CF-associated male infertility is especially relevant

given that the barrier to conception for men with CF, CAVD, or epididymal obstruction

has been overcome by advances made in the assisted reproductive technologies, thus

raising the concem of passing deleterious genetic traits ont0 subsequent generations [Mak

and Jarvi, 19961.

Alternative splicing variants other than exon 9- have been detected in CFTR

transcripts by RT-PCR [Bremer et al., 1992; Delaney et al., 19931. including ones which

involve the 5' end, portions of TM1, portions of NBD1, or the 3' end of the CFTR gene.

To date, these variants have not been dernonstrated to result in functional CFTR isoforms.

These patterns of alternative RNA splicing have been identified for the CFTR gene in a

variety of tissues, including lung, pancreas, intestine, and testis; however, similar

experiments have not been conducted in Wolffian-derived tissues. Since the latter

structures are highly sensitive to aberrant splicing as our study suggests, it would be

reasonable to assume that altematively spliced transcripts may occur with greater

fiequency in the reproductive tract. Therefore, we hypothesize that aberrantly spliced

transcripts other than exon 9-, previously discovered or novel, occur in the vas deferens

andor epididyrnis.

The tissue-specific difference in the splicing environment between the various

organs which express CFTR may be due to the presence of specific alternative splicing

factors. The identification and characterization of alternative splicing factors can be

approached by developing in viho splicing systems able to accurately reproduce cell-

specific splicing patterns. This should be possible either by using nuclear extracts fiom

the different ce11 types, or by complementing HeLa nuclear extracts [Smith et al., 1989;

Hodges and Bernstein, 19941. Other strategies for the isolation of factors include co-

injection of nuclei or nuclear extracts into Xenopur oocytes, dong with the experimental

transcnpt [Kelley and Perry, 1986; Kedes and Steitz, 19881. This system would also

vilru. With these approaches, alternative splicing factors which act on CFTR rnay be

identified and their mode of action subsequently characterized. The elucidation of

alternative splicing mechanisms could play an important role in development of novel

therapeutic strategies for CF. For example, it may be possible to design phmacologic

agents which upregulate the level andlor activity of constitutive splicing factors, or which

downregulate that of alternative splicing factors as means of augmenting levels of the

normal CFTR gene product.

Our findings may have a more immediate application to ow study population in

tems of providing prognostic information. Although too early in the post-operative

period for us to address this issue critically, it would be interesting to see whether a

correlation exists (after controlling for variables such as patient age, interval between

vasectomy and vasectorny reversal, and CF carrier statu) between the length of the IVS8-

T tract and the presence of sperm in the ejaculate pst-vasectomy reversal. Indeed, it

appears that azoospemiic men with idiopathic epididymal obstruction (who have a

significantly higher fiequency of the 5T variant (29%) compared to the general population

(5%) [Jarvi et al., 19951) have a lower rate of surgical success (def5ned as presence of

sperm in the ejaculate pst-vasoepididymostomy) compared to men with acquired

epididymal or vasal obstruction [Jarvi, K., unpublished data]. Although the difficulty of

surgery and the actual site of anastomosis are paramount in prognosticating post-

operative success, inadequate CFTR chloride function in the former group may contribute

to re-stenosis of the excurrent genital ducts.

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Organization, 19871

Semen volume:

Spenn count:

Percentage motility:

Percentage nonnal morphology:

White blood ce11 count:

'L'able 2. CFI'K gene mutations or vanations ana cHAvu

CFTR genotype 1 No. of CBAVD men (%y 1 Percentage of CBAVD mena

CFTR mutation / 1 43 1 CFTR mutation (19.8) CFTR mutation / -b 34

(1 5.7) CFTR mutation / 5T 85

5T / 5T -46% involve 5T variant

5T/-

- . .

-75% involve

CFTR gene

mutations

-1 8% have no CFTR gene mutations identified

aCalculated fiom a total of 217 CBAVD men [Chillon et al., 1995; Costes et al., 1995; Zielenski et

al., 1 995 b] . "'-" indicates no CFTR mutations detected.

general population

Group IVSS-T aflele frequency

9T 7T 5T

no. with allele / no. studied (%)

Present study 8/48 3 8/48 2/48 (48 chromosomes) (1 6.71~ (79.2)' (4.2)d

General population 571498 4 15/498 26/498 (498 chromosomes)a (1 1.5) (83.3) (5.2)

Data pooled from Kiesewetter et al., 1993, Cuppens et al., 1994, Dork et al., 1994, and

Chillon et al., 1995.

b x 2, 1 . 1 , p=0.28 for cornparison with the 9T allele fiequency in the generai population.

c x 2- -0.5, p=0.46 for cornparison with the 7T allele fiequency in the general population.

dp=0.75, Fisher's exact test, for comparison with the ST allele fiequency in the general

population.

Subject IVS8-T Nasal exon Vas Meanf SD MeankSD number genotype 9+ (Yo) deferens nasal vas deferens

exon 9+ (%) exon 9+ (%) exon 9+ (%)

aValues represent corrected proportions of exon 9+ transcripts, according to Fig. 35.

T tract allelesa

IVSS-T allele Proportion of nasal exon Pt Proportion of vasal exon 9+ ("/.) (%)

Values represent corrected proportions of exon 9+ transcripts, according to Fig. 35.

T tract genotypesa

IVS8-T genotype Proportion of nasal exon 9+ Proportion of vasal exon 9+ (%) (%)

'values represent corrected proportions of exon 9+ transcripts, according to Fig. 3J.

a' .SUI b a. 1 u)i S U - a VU u y u m j pj m ~ m m m m u m m m t - 8 ur- v a s u - U A r A\ 6 Y 1 4 - aUU - 4 - b . U 4 6 . V )i

splicing of CFTR exon 9. Three alleles have been identified in the polypy-rimidine tract

within the splice acceptor site of intron 8 of the CFTR gene: one with 9 thymidines, one

with 7 thymidines, and one with 5 thymidines (9T. 7T, 5T) [Chu et al., 19911. Al1 three

alleies are associated with two transcripts, one that is normally spliced with exon 9 intact

(exon 9+) and one that is aberrantly spliced, with skipping of exon 9 but i n - M e joining

of exons 8 to 10 (exon 99) [Chu et al., 1993 3. Transfection studies demonstrated that the

protein made from the exon 9- transcript is incapable of functioning as a CAMP-regulated

chloride channel [Strong et al., 1993; Delaney et ai., 19931. In addition, quantitative RT-

PCR analysis performed on branchial epithelial cells showed that individuals with the

longer T-tract length had a higher proportion of exon 9+ transcripts [Chu et al., 19931.

Therefore, CBAVD men with the 5T variant likely produce lower levels of normal CFTR

mRNA due to a less efficient splicing machinery.

V p I I L W 9 W U W r b V I a8bW GTGTG 7 A A A C A G 7T Of CFTR intron * GTGTG77777AAACAG 5T

CFTR 5'- 8 j gene I 3' -

/ Transcription

5 r l 3 E D 118 1 9 1 10 11 Exon 9- CFTR mRNA Exon 9+ CFTR mRNA

1 Translation 4 Inactive CFTR protein Functional CFTR protein

5Tl5T 5T17T 5T19T TTlTT R19T 9T19T IVS8-T genotype

Figure 1.

Figure 2. Detection of WS8-T genotype by oligonucleotide bybridization.

A) Leukocy-te genomic DNA fiom study subjects were extracted and the region

encompassing the intron 8 polypyrimidine tract was amplified by PCR with

primers indicated in the schematic to generate a predicted product of 560 bp.

B) Amplification was evaluated by ethidium bromide staining and gel

electrophoresis. PCR product was generated fiom al1 subjects (numbered 1 to

24) (M=100 bp marker; Neg==no DNA template). C) An aliquot of amplified

DNA was denatured and vacuum-blotted to nylon membrane and hybridized with

radiolabeled oligonucleotides which specifically recognize ST, 7T, or 9T aleles.

Autoradiography revealed that subjects 1, 9, and 23 possess the 7T/9T, 7T/7T,

and 5T/7T genotypes, respectively.

poly-T tract

CFTR 5' 1 8 1 gene 3'

Figure 2A.

Figure 2B.

Subject number

1 9 23

probe TI 9 5G-GTGTGTTTllTAACAG-31 Probe sequence

TTIST 7TffT ST/7T

NS8-T genotype

Figure 2C.

type (WT) CFTR cDNA (exon 9+) were subjected to two rounds of nested PCR with

primers as shown. Amplified products were assessed by ethidium bromide staining and

agarose gel electrophoresis (Neg controkno DNA). Second round PCR fragments (834

bp) were extracted fiom the gel and purified. B) Fragments of CFTR exon 9- transcripts

(651 bp) were obtained in a similar marner from human lung total RNA. C) Sarne

volumes of the isolated exon 9+ and exon 9- fragments were loaded ont0 separate lanes

and size fractionated by agarose gel electrophoresis for cornparison. Note that a higher

yield was obtained for the exon 9+ hgments, as reflected by their greater intensity under

ultraviolet light. D) To obtain equivalent concentrations of exon 9+ and exon 9-

transcripts, the exon 9+ fragments were serially diluted and mixed with a constant volume

of the exon 9- frsigments, and evaluated by Southem analysis. Hybridization with an

exon 10 specific probe (C16B) produced bands of the two kgments, and quantification

by optical density (OD) measurements demonstrateci that the concentration of the isolated

exon 9+ fragments was -1.82 times that of exon 9- fragments. Thus, the exon 9+

fi-agments were diluted 1.82 tirnes, making them equivalent in concentration to the exon

9- fragments. E) In order to ven@ calculations and the purity of the exon 9+ and exon 9-

isolates, Southem analysis and hybridization with a common probe (C16B), exon 9+

specific probe (9i-Ss), and exon 9- specific probe (ex8/10) were used. F) Hybridization

with C 16B and OD measurements confinned that concentrations of exon 9+ (OD=2.482)

and exon 9- (OD=1.478) fragments were equivalent. Hybridization with 9i-5s and ex8/10

validated the purity of the isolates, as s h o w by their annealing to only the exon 9+ and

exon 9- fkgrnents, respectively. G-1) The exon 9+ and exon 9- fragments were serially

subjected to the same PCR conditions and quantification procedure as for the nasal and

vas deferens specimens. The experimental proportions of exon 9+ and exon 9- transcripts

were evaluated by scanning densitometry, with the sum of OD measurements for exon 9+

and exon 9- taken as 100%. J) Graph of actual versus experimental proportions of CFTR

exon 9+ transcripts. Al1 experimental proportions of nasal and vas deferens exon 9+

mRNA were adjusted according to this graph.

Figure 3C.

Vol (pl) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0

Figure 30.

Figure 3E.

Exon 9- fragments

c c e c c c r e r O O s d a w 8 Y 8

w l u w

7 1 8 1 1 0 ( 11 1 1 2 651bp

Exon 9+ OD 1.482

Exon 9- OD 1.478

Figure 3F.

Exon 9- fragments

Figure 36.

Exon 9+ fragments

Exon 9- fragments

Figure 3H.

Figure 31.

Figure 35.

Figure 4. Evaluation of RNA extraction and cDNA synthesis. Total RNA

was extracted fiom nasal and vas deferens specimens, converted to cDNA, and

PCR amplified for a region of the GAPDH transcript. The products were

analyzed by gel electrophoresis, Southern blotted, and hybridized with a 32P-

labeled GAPDH probe specific for the amplified region. The resuitant

autoradiographic bands were quantified by densitometric scanning. Differences

in the yield of RNA extraction and cDNA synthesis were adjusted for, prior to

amplification with CFTR primers. For example, the concentration of cDNA

obtained from the vas of subject 5 was -3.4 times l e s than that from subject 1,

hence 3.4 times the amount of cDNA from subject 5 was used for CFTR mRNA

analy sis.

Vas sample no.: I 2 3 4 5 6 7 8

OD:

Figure 4.

Figure 5. Evaluation of CFTR mRNA. A) Total RNA was extracted fiom

nasal and vas deferens samples, converted to cDNA, and PCR amplified using

primers fiom exons 8 and 10 (X8-5 and C16D, respectively), producing two

different sized frzigments which corresponded to the exon 9+ (416 bp) and exon

9- (233 bp) transcripts. B) The CFTR cDNA products were then separated by

agarose gel electrophoresis and transferred to nylon memebrane by Southem

blotting, followed by hybridization with 32P-labeled probes specific for the exon

9+ or exon 9- fragments (9i-5s and ex8110, respectively) and one common to

both (C 16B). C) C 16B, a sequence within exon 10, identified two species (lane

1). The exon 9+ specific probe (9i-5s) detected only the larger transcript,

corresponding to CFTR mRNA with exon 9 intact (Xane 2), while the exon 9-

specific probe (ex8/10) detected only the smaller transcript (lane 3) which

represented the i n - h e deleted exon 9 message.

Exon Q + ~ R N A 1 8 1 9 1 10 1 * -

X l b C16D - 416 bp

Exon s - mRNA lIia3rl - -

Xô-S Cl60

Figure SA.

Exon 9+ 1 ~ R N A 8 1 9 1 10 1416bp = 913s

Exon 9- mRNA

Figure 5B.

Figure 5C.

Figure 6. Quantification of nasal and vas deferens CFTR m W A . The

density of cDNA fragments on the autoradiographs was measured by scanning

densitometry. The proportions of nasal and vas deferens exon 9+ mWA, as a

percentage of total CFTR transcripts, were determined by using the total

densitometric units of both transcripts as 100%.

I 7T19T 1 7Tff T 1 5 ~ 1 7 ~ 1 Sublect no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Figure 6.

Figure 7. Relationship between CFïR IVS8-T genotype and proportion of

exon 9+ transcripts in nasal epitheiia (*) or vas deferens (O). Each specimen

is represented by a separate symbol. Horizontal bars indicate mean values.

Vas Nasal Via8 Nasal V Nasal

5 T M 7TRT 7T19T

Figure 7.

Figure 8. Cornparison of the proportion of C F ï R exon 9+ trsnscnpts

within nasal epithelia and vas deferens from the same subject. Intra-

individuai values are c o ~ e c t e d by straight lines.

Nasal Figure 8.

Vas

Figure 9. Influence of CFTR genotype on phenotype by the proportion of

normal CM.R mRNA. A typical patient with CBAVD rnay have a severe

CFTR gene mutation (e.g., AF508) in one CFTR allele and the less efficiently

splicing 5T variant in the other. He may produce a suficient proportion of

normal (exon 9+) CFTR mRNA (and therefore protein) fiom the 5T ailele in the

lung (32% of total CFTR transcripts) to confer a normal phenotype but not so in

the reproductive tract (26%), thus giving him the isolated absence of vas deferens

phenotype without any pulmonary manifestations. In contrast, a typical CF

carrier may have a severe CFTR mutation in one copy of the CFTR gene but the

more eficiently splicing 7T variant in the other chromosome. Although he will

produce less of the normal CFTR mRNA in the reproductive tract (38%)

comparecl to the lung (41%) fiom the 7T allele, lmth proportions exceed the

threshold for normalcy, thus giving him a completely normal phenotype. The

threshold level for normal/abnomal phenotype depicted in the figure is arbitrary

and may vary between different tissues.

Congenital 811. Absence of Vas Deferens Cystic Fibrosis Camer

CFTR mut IlF cFTRmu/7T

1 :g

nomulphmnotyp. H . . . . . . . . . . . . . . . . . . . rbnomul ph- g 4kKmnrl phmnotyp.

;ii 1: &

Lung WdCRan Lung Wolffian

Figure 9.

APPLIED A IMAGE. lnc 1653 East Main Street - -. - - Rochester, NY 14609 USA -- -- - - Phone: il 61482-0300 -- -- - - Fax: 7161288-5989

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