## Abstract

Microdeletions of Yq are associated with azoospermia and severe oligozoospermia. In general, men with deletions are infertile and therefore deletions are not transmitted to sons unless in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are performed. We report an unusual family characterized by multiple members with infertility and Yq microdeletion. Complete reproductive history, semen analyses and blood samples were elicited from relevant family members. DNA preparation and quantification were performed using commercial kits. A total of 27 pairs of sequence tagged sites based primer sets specific for the Y microdeletion region loci were used for screening. Southern blots using deleted in azoospermia (DAZ) and ribosomal binding motif (RBM) cDNAs were then analysed for confirmation. The proband, his three brothers and father were all found to be deleted for DAZ but not RBM. At the time of analysis, the proband's father was azoospermic whereas his four sons were either severely oligozoospermic or azoospermic. Unlike their father, the four sons are infertile and have no offspring, except for one of them who achieved a daughter only after IVF/ICSI treatment for infertility. Microdeletions of Yq involving the DAZ gene are associated with a variable phenotypic expression that can include evidently normal fertility.

## Introduction

Infertility occurs in ~14% of couples (Mosher, 1985) and abnormalities in the male partner are estimated to be present in up to half of the cases (Swerdloff et al., 1985). Efforts to evaluate the causes of azoospermia have shown that after exclusion of traditionally recognizable causes (i.e. abnormal karyotype, obstruction, varicocoele, hormonal defect, etc.), most cases (50–75%) are unexplained and are termed idiopathic (Pryor et al., 1997). Recently, it has been reported that up to 30% of men with idiopathic' azoospermia have microdeletions of the Y chromosome (Henegariu et al., 1993; Ma et al., 1993; Nagafuchi et al., 1993; Kobayashi et al., 1994; Najmabadi et al., 1996; Reijo et al., 1996; Vogt et al., 1996; Pryor et al., 1997). Exactly how and whether or not these microdeletions cause azoo/oligozoospermia is the subject of both intense investigation and debate.

Essential to the argument that Y microdeletions cause infertility is the observation that fertile men rarely manifest Y microdeletions. Microdeletions in four out of 200 fertile men studied have been reported (Pryor et al., 1997). However, the deletions in these men were very small and most likely represented insignificant polymorphism. Relatively large deletions of the kind associated with male infertility have not been reported in men with normal fertility. Although it is generally assumed that these deletions arise de novo and that father to son transmission of Y microdeletion would not be expected, a few rare instances of father to one son transmission of Y chromosome microdeletion have been reported (Kobayashi et al., 1994; Stuppia et al., 1996; Vogt et al., 1996; Pryor et al., 1997). However, vertical transmission of a microdeletion involving the deleted in azoospermia (DAZ) locus from father to one son has been reported in only three cases (Kobayashi et al., 1994; Vogt et al., 1996; Pryor et al., 1997). We now describe a four-generation family in which an azoospermic father and his four infertile sons all share an apparently identical microdeletion that includes the DAZ locus. This family represents the first and only report of spontaneous vertical transmission of DAZ deletion to multiple offspring. It provides evidence that a single Yq microdeletion can result in varying phenotypic expression in different individuals. It is clinically significant, in that the presence of a microdeletion is not an absolute marker for infertility and can be associated with apparently normal fertility.

## Materials and methods

Screening for Yq microdeletion was performed on a routine basis for male infertility using a protocol reviewed and approved by the Institutional Review Board of College of Physicians & Surgeons, Columbia University. Samples were taken from patients after informed consent.

### Semen analysis

Results were analysed using WHO criteria with a Nikon phase contrast microscope.

### Serum hormone concentrations

Follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were measured by solid-phase, two site chemiluminescent enzyme immunometric assay (Immulite; Diagnostic Products Corporation, Los Angeles, CA, USA). Normal ranges for men are FSH <10 mIU/ml; LH <10 mIU/ml; and testosterone 270–1070 ng/dl.

### Genomic DNA

Extraction of genomic DNA from whole blood was performed by lysis of red blood cells, followed by lysis of white blood cells and their nuclei. Cellular proteins were removed by salt precipitation, and genomic DNA was precipitated with isopropanol using Puregene DNA extraction kit (Gentra Systems, Inc. Minneapolis, MN, USA; catalogue no. D-5004).

### Polymerase chain reaction (PCR)

Primers were produced as dried oligonucleotides on an automated DNA synthesizer (Perkin-Elmer Applied Biosystems Inc., Foster City, CA, USA). A total of 27 Y chromosome specific sequence tagged sites (STS) (Figure 1) were selected from an STS map (Vollrath et al., 1992). They include the three proposed spermatogenesis loci AZFa, AZFb, and AZFc (as per Vogt et al., 1996) spanning Yq intervals 5, 6 and 7. As a rapid screening protocol, a PCR multiplex system composed of two to six different primer pairs was used in a total of six multiplexed reactions (Table I). With each PCR run, a female control and a normal male control were included. All PCR reactions were run in polycarbonate (Techne®) plates in an MJ Research® machine. The PCR conditions were essentially as previously described (Henegariu et al., 1993). Briefly, in a 14 μl total volume reaction, 50 ng of genomic DNA was used as template, 1 μl of primer standard solution (mix I or II or III or IV or V or VI consisting of 10 pmol per primer), 12 μl of PCR cold mix' (1.5 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 5% DMSO, 1× Taq polymerase reaction buffer without Mg2+), 1.25 IU Taq DNA polymerase (Promega) and 1 drop of oil. The complete mixes were placed directly in a thermocycler preheated to 94°C. Cycling conditions for 27 cycles were: 94°C, 30 s (melting); 55°C, 45 s (annealing); and 72°C, 60 s (extension). The final extension time was 5 min. The PCR reaction products were then separated on 3% agarose gels (Bio-Rad, ultra-pure grade) by electrophoresis in TBE buffer. PCR products were stained with ethidium bromide and visualized by exposure to ultraviolet light. STS showing no amplification in multiplex reactions were confirmed by single reaction PCR with appropriate positive and negative controls. An STS was considered to be absent after three amplification failures.

### Southern hybridization

Southern blotting was performed according to established protocol (Sambrook et al., 1989). Briefly, 5 μg genomic DNA was digested with HindIII or TaqI, run on a 0.7% agarose gel in standard TBE buffer, transferred to a nylon membrane, and hybridized with 32P-labelled probes. The DAZ probe was the purified insert of a plasmid (pDP1577) containing the full length cDNA (Reijo et al., 1995). Similarly, the RBM probe was the plasmid insert of an RBM cDNA clone (MK5) (Ma et al., 1993).

### Paternity determination

Paternity of all four sons was confirmed by showing the expected segregation of four highly polymorphic autosomal markers (Weber and May, 1989). These were D21S156, D21S270, D13S132, and D13S159 with heterozygosities of 0.83, 0.86, 0.84 and 0.90 respectively.

### Fluorescent in-situ hybridization (FISH)

FISH for DAZ was performed with Cosmid 63C9 (Saxena et al., 1996), using established methods (Yu et al., 1996).

## Results

The finding that individual II-8 in the pedigree was azoospermic but did not have a microdeletion was a surprise. The DAZ locus in this individual was further tested by Southern analysis using the DAZ cDNA and a different restriction enzyme (TaqI). It failed to show any abnormality of the DAZ locus. In addition, FISH with the DAZ Cosmid 63C9 showed normal intensity (data not shown).

The proband (III-8) and his older brother (III-6) were seeking infertility treatment. After extensive counselling, they opted for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The proband (III-8) and his wife (III-9) underwent two IVF-ICSI cycles that failed to produce a pregnancy secondary to poor ovarian response. The proband's brother (III-6) and his wife (III-7) underwent one cycle of controlled ovarian hyperstimulation and nine oocytes were retrieved. Eleven mature spermatozoa were found in three ejaculates on the day of retrieval and used for ICSI. Three oocytes fertilized which subsequently cleaved and were transferred. She delivered a healthy female baby (IV-2).

## Discussion

We report an exceptional family in which an azoospermic father and his four infertile sons share an apparently identical Yq microdeletion involving the DAZ locus. The de-novo mutation that led to the microdeletion of DAZ appears to have originated in the proband's father (II-1). This deletion is expected to cause azoo/oligozoospermia and male infertility, and yet he spontaneously conceived five children and was unaware of any fertility problem. Interestingly, however, all four sons are infertile and are either azoospermic or severely oligozoospermic.

This family raises several issues with regards to the association between Yq microdeletion and infertility. First, it confirms that vertical transmission of Yq microdeletion is possible and can lead to subsequent infertility in the male offspring. Second, it is obvious that the same deletion can result in different phenotypes in different individuals. Although the father (II-1) of the four boys in this family was azoospermic at the time of analysis, he fathered his first child at the age of 25 and his last one at the age of 38 years. Thus, he possessed some degree of fertility over a large span of years. Likewise, microdeletion of the Y chromosome, specifically DAZ, does not necessarily imply a lifelong history of azoospermia nor does it preclude the formation of a large family. His four sons, on the other hand, are infertile and either azoospermic or severely oligozoospermic.

The DAZ gene has been proposed as the azoospermia factor on the Y chromosome. This family shows clearly that while DAZ may have a critical role in spermatogenesis, it is not essential for fertility. Furthermore, total loss of the DAZ gene cluster can be associated with a histological picture of Sertoli cell only' as well as sperm maturation arrest (Foresta et al., 1997; Pryor et al., 1997). Several authors have found a poor correlation between the location of Y microdeletions (including DAZ deletions) with the clinical and histological phenotype of the patients (Reijo et al., 1995, 1996; Vogt et al.; 1996; Silber et al., 1998). The findings in this family agree that such a correlation may turn out to be quite problematic. Testicular biopsy of the proband's brother (III-6) showed a picture of Sertoli cell only' whereas the proband (III-8 with sperm count 0.5×106/ml) clearly would be expected to have some degree of sperm maturation on biopsy. Furthermore, testicular biopsy may not be representative of the entire testicle because there may be geographic heterogeneity for spermatogenesis as in individual III-6 whose ejaculates contained mature spermatozoa.

We can only speculate about the basis for phenotypic differences between family members with the same deletion. It is well known that identical deletions within autosomes may result in different phenotypes (Schinzel, 1994). One can postulate that such differences are consequences of each individual's exposure to his environment or expression of various modifying genes. A fertile father has been described with a microdeletion that widened when transmitted to his infertile son (Stuppia et al., 1996). Although variable extensions at the borders of the deletion may exist between our different family members, these molecular extensions cannot be distinguished by interval mapping. By PCR analysis, the same STSs failed to amplify in our five individuals and Southern hybridization with the DAZ probe confirmed a complete deletion of this gene cluster. Although it is possible that the deletions observed are, in fact, not identical and adjacent areas may contain important genes that modulate the degree of phenotypic expression, these results still indicate a large overlap of deleted Y DNA (including the loss of DAZ gene cluster) in each individual of this unique family.

We were fascinated by the fact that the proband's uncle (II-8) has infertility and azoospermia but no apparent microdeletion by STS testing. Since southern blot analysis using both the RBM and DAZ probes as well as FISH analyses using a DAZ cosmid were all entirely normal, we are forced to conclude he has a different aetiology underlying his infertility. Admittedly, it is possible that he may have a smaller or point mutation/perturbation or proximal/distal rearrangement that is not detectable by current methods. He gave no history of exposure to gonadotoxins or other definable factors that were likely to affect spermatogenesis.

Until recently, Y microdeletion has had little clinical significance, since a man with a deletion will not, in general, reproduce. However, utilizing ICSI and testicular sperm aspiration (TESA), combined with IVF, it is now possible for oligo/azoospermic men with Y microdeletion to achieve pregnancies (Mulhall et al., 1997; Silber et al., 1998, and individual III-6). This has fostered concerns that such pregnancies may produce male offspring with similar microdeletions and subsequent infertility (Reijo et al., 1996; Girardi et al., 1997; Kremer et al., 1997). Indeed, Yq microdeletion can be transmitted to male offspring via ICSI (Kent-First et al., 1996). The family we report suggests that men with Yq microdeletions (such as individual II-1) who achieve pregnancies will transmit the same microdeletion and the risk of infertility to their sons (individuals III-1, III-4, III-6, III-8). Therefore, patients should be offered Y microdeletion screening prior to ICSI and they should be counselled on the certainty of transmitting the Yq microdeletion and possibly infertility to their sons. As more research is focused on genetic aetiologies of male infertility, identification of genes involved in spermatogenesis should provide insight into the pathophysiology of male infertility and a more rational basis for initiating therapy.

Table I.

Multiplex polymerase chain reaction (PCR) scheme used for the 27 STS primer pairs. The primers are ordered by decreasing expected lengths

Multiplex mix Sequence tagged site (STS) Expected PCR product length (bp) Corresponding locus
157 285 DYS240
154 245 DYS238
142 196 DYS230
145 160 DYF51S1
131 143 DYS222
139 120 DYS227
II 134 301 DYS224
136 235 DYS226
129 194 DYS220
132 159 DYS7
152 125 DYS236
III 143 311 DYS231
55 256 DYF67S1
130 173 DYS221
149 132 DYS1 (DAZ
147 100 DYS232
IV  83 275 DYS11
158 231 DYS241
148 202 DYS233
138 170 DYF49S1
153 139 DYS237
164 690 DYF65S1
84 326 DYS273
87 252 DYS275
144 143 DYF50S1
VI 159 550 DYZ2
160 236 DYZ1
Multiplex mix Sequence tagged site (STS) Expected PCR product length (bp) Corresponding locus
157 285 DYS240
154 245 DYS238
142 196 DYS230
145 160 DYF51S1
131 143 DYS222
139 120 DYS227
II 134 301 DYS224
136 235 DYS226
129 194 DYS220
132 159 DYS7
152 125 DYS236
III 143 311 DYS231
55 256 DYF67S1
130 173 DYS221
149 132 DYS1 (DAZ
147 100 DYS232
IV  83 275 DYS11
158 231 DYS241
148 202 DYS233
138 170 DYF49S1
153 139 DYS237
164 690 DYF65S1
84 326 DYS273
87 252 DYS275
144 143 DYF50S1
VI 159 550 DYZ2
160 236 DYZ1
Table II.

Semen analyses and hormone profiles of relevant family members

ID Relationship to proband Age (years) Sperm count (×106/ml) FSH (mIU/ml) LH (mIU/ml) Testosterone (ng/dl)
FSH = follicle stimulating hormone; LH = luteinizing hormone; NA = not analysed.
III-8 Proband 24  0–0.5  3.5  4.5 485
III-6 Brother 33  3 spermatozoa  5.1  2.5 279
III-4 Brother 37  0.1  5.5  1.7 499
III-1 Brother 38 NA  6.3  1.6 414
II-1 Father 63 21.2  3.3 392
II-8 Uncle 44 40.7  8.7  37
Normal values >20 <10.0 <10.0 270–1070
ID Relationship to proband Age (years) Sperm count (×106/ml) FSH (mIU/ml) LH (mIU/ml) Testosterone (ng/dl)
FSH = follicle stimulating hormone; LH = luteinizing hormone; NA = not analysed.
III-8 Proband 24  0–0.5  3.5  4.5 485
III-6 Brother 33  3 spermatozoa  5.1  2.5 279
III-4 Brother 37  0.1  5.5  1.7 499
III-1 Brother 38 NA  6.3  1.6 414
II-1 Father 63 21.2  3.3 392
II-8 Uncle 44 40.7  8.7  37
Normal values >20 <10.0 <10.0 270–1070
Figure 1.

Y chromosome map and microdeletions in subinterval 6D–6F of the Y chromosome long arm in the proband (III-8), his father (II-1) and three brothers (III-1, III-4, III-6). The presence of a sequence tagged site (STS) is indicated by the solid portion of the column. The STS not amplified are marked with asterisks. The approximate boundaries of AZFa, AZFb, and AZFc regions (as per Vogt et al., 1996) are shown.

Figure 1.

Y chromosome map and microdeletions in subinterval 6D–6F of the Y chromosome long arm in the proband (III-8), his father (II-1) and three brothers (III-1, III-4, III-6). The presence of a sequence tagged site (STS) is indicated by the solid portion of the column. The STS not amplified are marked with asterisks. The approximate boundaries of AZFa, AZFb, and AZFc regions (as per Vogt et al., 1996) are shown.

Figure 2.

Pedigree of the four-generation family with results of Yq microdeletion testing. The proband (III-8) is indicated by an arrow. The proband (III-8) is severely oligozoospermic and microdeleted for subinterval 6D-6F of Yq. His father (II-1) was found to be azoospermic and the two brothers (III-4, III-6) were severely oligozoospermic. The third brother (III-1) declined semen testing. The proband, his father and three brothers were all found to have an apparently identical microdeletion including DAZ. The proband's uncle (II-8) was found to have azoospermia but no Yq microdeletion was detected.

Figure 2.

Pedigree of the four-generation family with results of Yq microdeletion testing. The proband (III-8) is indicated by an arrow. The proband (III-8) is severely oligozoospermic and microdeleted for subinterval 6D-6F of Yq. His father (II-1) was found to be azoospermic and the two brothers (III-4, III-6) were severely oligozoospermic. The third brother (III-1) declined semen testing. The proband, his father and three brothers were all found to have an apparently identical microdeletion including DAZ. The proband's uncle (II-8) was found to have azoospermia but no Yq microdeletion was detected.

Figure 3.

Southern blot with DAZ probe. The entire DAZ locus in individuals II-1, III-8, and III-6 is absent, whereas it appears to be present and normal in the control male and individuals I-1, II-8 and IV-1.

Figure 3.

Southern blot with DAZ probe. The entire DAZ locus in individuals II-1, III-8, and III-6 is absent, whereas it appears to be present and normal in the control male and individuals I-1, II-8 and IV-1.

Figure 4.

(a) Metaphase from individual I-1 (control) after FISH using probe DYZ3 (ONCOR) to identify the Y centromeric DNA (green) and probe Cosmid 63C9 to identify the DAZ-containing chromosome region (red). Both signals are seen on the Y chromosome. (b) Metaphase from individual II-1 using the same probes. Only the green signal is seen, identifying the Y chromosome, but no signal for DAZ region is present.

Figure 4.

(a) Metaphase from individual I-1 (control) after FISH using probe DYZ3 (ONCOR) to identify the Y centromeric DNA (green) and probe Cosmid 63C9 to identify the DAZ-containing chromosome region (red). Both signals are seen on the Y chromosome. (b) Metaphase from individual II-1 using the same probes. Only the green signal is seen, identifying the Y chromosome, but no signal for DAZ region is present.

1
To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, Division of Reproductive Endocrinology, College of Physicians & Surgeons, Columbia University, 622 West 168th Street, PH 16–28, New York, NY 10032, USA

We thank the families for their cooperation in the study; Dr David C.Page for providing the DAZ cDNA probe and his invaluable help with this manuscript; Dr Kun Ma for providing the MK5 (RBM1) cDNA probe; Dr Peter Vogt for the DNA of Yq microdeleted individuals used to validate our STS PCR methodology; and C.C.Yu and Patricia Lanzano for their invaluable technical assistance.

This study was funded in part by the Columbia Presbyterian Medical Center Office of Clinical Trials House Staff Awards.

## References

Foresta, C., Ferlin, A., Garolla, A. et al. (
1997
) Y-chromosome deletions in idiopathic severe testiculopathies.
J. Clin. Endocrinol. Metab.
,
82
,
1075
–1080.
Girardi, S.K., Mielnik, A. and Schlegel, P.N. (
1997
) Submicroscopic deletions in the Y chromosome of infertile men.
Hum. Reprod.
,
12
,
1635
–1641.
Henegariu, O., Hirschmann, P., Kilian, K. et al. (
1993
) Rapid screening of Y chromosome in idiopathic sterile men, diagnostic for deletions in AZF, a genetic Y factor expressed during spermatogenesis.
Andrologia
,
26
,
97
–106.
Kent-First, M.G., Kol, S., Muallem, A. et al. (
1996
) The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers.
Mol. Hum. Reprod.
,
2
,
943
–950.
Kobayashi, K., Mizuno, K., Hida, A. et al. (
1994
) PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis.
Hum. Mol. Genet.
,
3
,
1965
–1967.
Kremer, J.A.M., Tuerlings, J.H.A.M., Meuleman, E.J.H. et al. (
1997
) Microdeletions of the Y chromosome and intracytoplasmic sperm injection: from gene to clinic.
Hum. Reprod.
,
12
,
687
–691.
Ma, K., Inglis, J.D., Sharkey, A. et al. (
1993
) A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling human spermatogenesis.
Cell
,
75
,
1287
–1295.
Mosher, W.D. (
1985
) Reproductive impairments in the United States, 1965–1982.
Demography
,
22
,
415
–430.
Mulhall, J.P., Reijo, R., Alagappan, R. et al. (
1997
) Azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection.
Hum. Reprod.
,
12
,
503
–518.
Nagafuchi, S., Namiki, M., Nakahori, Y. et al. (
1993
) A minute deletion of the Y chromosome in men with azoospermia.
J. Urol.
,
150
,
1155
–1157.
Najmabadi, H., Huang, V., Yen, P. et al. (
1996
) Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence tagged site-based mapping strategy.
J. Clin. Endocrinol. Metab.
,
81
,
1347
–1352.
Pryor, J.L., Kent-First, M., Mulhallem, A. et al. (
1997
) Microdeletions in the Y chromosome of infertile men.
N. Engl. J. Med.
,
336
,
534
–539.
Reijo, R., Lee, T.Y., Salo, P. et al. (
1995
) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene.
Nature Genet.
,
10
,
383
–393.
Reijo, R., Alagappan, R.K., Patrizio, P. et al. (
1996
) Severe oligospermia resulting from deletions of azoospermia factor gene on Y chromosome.
Lancet
,
347
,
1290
–1293.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
Saxena, R., Brown, L.G., Hawkins, T. et al. (
1996
) The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned.
Nature Genet.
,
14
,
292
–299.
Schinzel, A. (1994) In Epstein, C. (ed.), The Phenotypic Mapping of Down Syndrome and Other Aneuploid Conditions. Wiley-Liss, NewYork, pp. 19–32.
Silber, S.J., Alagappan, R., Brown, L.G. et al. (
1998
) Y chromosome deletions in azoospermic and severely oligozoospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction.
Hum. Reprod.
,
13
,
3332
–3337.
Stuppia, L., Calabrese, G., Franchi, P.G. et al. (
1996
) Widening of a Y-chromosome interval-6 deletion transmitted from a father to his infertile son accounts for an oligozoospermia critical region distal to the RBM1 and DAZ genes.
Am. J. Hum. Genet.
,
59
,
1393
–1395.
Swerdloff, R.S., Overstreet, J.W., Sokol, R.Z. et al. (
1985
) Infertility in the male.
Ann. Int. Med.
,
103
,
906
–919.
Vogt, P.H., Edelmann, A., Kirsch, S. et al. (
1996
) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11.
Hum. Mol. Genet.
,
5
,
933
–943.
Vollrath, D., Foote, S., Hilton, A. et al. (
1992
) The human Y chromosome: 43 interval map based on naturally occurring deletions.
Science
,
258
,
52
–59.
Weber, J.L. and May, P.E. (
1989
) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction.
Am. J. Hum. Genet.
,
44
,
388
–396.
Yu, F., Warburton, D., Wellington, D. et al. (
1996
) Assignment of gene coding for alpha2 subunit of soluble guanylyl cyclase to position 11q21–22 on human chromosome 11.
Genomics
,
33
,
334
–336.