Submicroscopic terminal deletion of 1p36.3 and Xp23 hidden in complex chromosome rearrangements: independent mechanism of telomere restitution on the two chromatids

In this study, we report two cases each with a complex chromosome rearrangement concealing a submicroscopic terminal deletion. The first case had a mos 46,XX,der(1)t(1;9)(p36.3;p13). ish der(1)(wcp9 +, 1ptel-, 9ptel +, pan tel +)[88]/46,XX. ish del(1)(1ptel -, 9ptel -, pan tel +)[12] karyotype. Scrutiny by FISH using wcp 9, 1ptel, 9ptel, and pan telomeric probes found a subtelomeric 1ptel deletion on the der(1) in the abnormal cell line and on a chromosome 1 in the apparently normal cell line. The telomere (TTAGGG)n, however, was present on the terminal ends of both copies of chromosome 1 in the apparently normal and abnormal cell lines. The second case had a de novo mos 46,X,der(X)t(X;22)(p22.3;q11.2),inv dup(22)(q11.2).ish der(X)(wcpX +,wcp22 +,KAL +, STS -,Xptel -,BCR +),inv dup(22)(wcp22 +,TUPLE ++,BCR -)[85]/45,X,der(X)t(X;22)(p22.3;q11.2),- 22[15].ish der(X)(wcpX +,wcp22 +, KAL +,STS -,Xptel -,BCR +) karyotype. FISH probes identified a terminal Xpter deletion, distal to the KAL gene. The two rearrangements are hypothesized to have been initiated by a terminal deletion. We propose a model for the formation of the rearrangement in Case 1, which invokes independent telomere stabilization of the sister chromatids. A terminal deletion 1pter in meiosis, was followed by acquiring or regenerating a telomere (TTAGGG)n cap on one chromatid and the other chromatid was involved in a translocation with a chromosome 9 chromatid. Following segregation of this chromosome the viable cell line survives to form the mosaic karyotype. Our findings suggest that subtelomeric deletions should be ruled out in cases with complex and simple rearrangements involving the terminal regions.     

Amplification of C-MYC as the origin of the homogeneous staining region in ovarian carcinoma detected by micro-FISH.

Homogeneous staining region (hsr), a cytogenetic indicator of gene amplification, has been frequently found in ovarian carcinoma (ovc). To identify the origin of the hsr, chromosome microdissection combined with polymerase chain reaction and fluorescence in situ hybridization (FISH) was applied to two human ovarian cancer cell lines, GR and MLS/P. The hsr probes were labeled with biotin or digoxigenin and hybridized to normal metaphase spreads to elucidate the chromosomal origin and regional localization of the amplified genes. FISH to normal metaphase spreads with the probe generated from the whole hsr-bearing chromosome from GR hybridized to 8q24, 2p13-->2q11.2, 10pter-->10p15, 10p12-->10q11.2, 5q23-->5q31, and 5q33-->5qter. For MLS/P, the hsr-bearing marker chromosome hybridized to 8q and 15q. In both cases, detailed FISH analysis revealed enhanced signal intensity at the 8q24 locus, which coincides with the chromosomal location of the C-MYC oncogene. To verify the involvement of C-MYC in hsr formation, in situ hybridization with a probe specific for the C-MYC oncogene was conducted and confirmed the amplification of C-MYC as the origin of the hsr. The whole hsr-bearing chromosome for GR is designated as rev ish der(10) (10pter-->10p15::8q24hsr:: 10p12-->10q11.2::8q24::2q11.2-->2p13::2p13 -->2q11.2::8q24::10q11-->10p11.2:: 5q23-->5q31::5q33-->5qter (wcp10+,D10Z1++,wcp2+,D2Z++,wcp5+,wcp8+ ,C-MYC++/hsr). The hsr-bearing marker for MLS/P is designated as rev ish der(8)(qter-->8q24::8q24::8q24-->8q10:: 8q10-->8q24::8q24::8q24-->8qter:: 15q11-->15qter)(wcp8+, D8Z1+,wcp15+,C-MYC++. FISH with the probe generated from the hsr of GR also painted the hsr in MLS/P, indicating that the two hsrs have shared homology, which indicates that the amplification of 8q24/C-MYC as the origin of hsr may be a nonrandom genomic alteration in ovc.     

Cryptic subtelomeric translocations in the 22q13 deletion syndrome

Cryptic subtelomeric rearrangements are suspected to underlie a substantial portion of terminal chromosomal deletions. We have previously described two children, one with an unbalanced subtelomeric rearrangement resulting in deletion of 22q13→qter and duplication of 1qter, and a second with an apparently simple 22q13→qter deletion. We have examined two additional patients with deletions of 22q13→qter. In one of the new patients presented here, clinical findings were suggestive of the 22q13 deletion syndrome and FISH for 22qter was requested. Chromosome studies suggested an abnormality involving the telomere of one 22q (46,XX,?add(22)(q13.3)). FISH using Oncor D22S39 and Vysis ARSA probes confirmed a terminal deletion. A multi-telomere FISH assay showed a signal from 19qter on the deleted chromosome 22. Results were confirmed with 19qtel and 22qtel specific probes. The patient is therefore trisomic for 19qter and monosomic for 22qter. The patient''s mother was found to have a translocation (19;22)(q13.42;q13.31). We also re-examined chromosomes from two patients previously diagnosed with 22q deletions who were not known to have a rearrangement using the multi-telomere assay. One of these patients was found to have a derivative chromosome 22 (der(22)t(6;22)(p25;q13)). No evidence of rearrangement was detected in the other patient. Thus we have found the 22q13 deletion to be associated with a translocation in three of four patients. This report illustrates the usefulness of examining patients with hypotonia, severe language delay, and mild facial dysmorphism for this syndrome and suggests that most of these deletions may be unbalanced subtelomeric rearrangements.     

Unique karyotypes in two patients with Prader-Willi syndrome

A physical disruption of the Prader-Willi syndrome (PWS) chromosome region is thought to cause PWS. We describe 2 girls with PWS phenotype, who had unique chromosome 15 abnormalities. The first patient showed mosaicism: 45,XX,t(15;15)(qter→p11.1::q11.200→ qter)/46,XX,t(15;15)(qter → p11.1::q11.200→ qter), + mar. The band 15q11.2 apparently remained intact in the t(15;15) chromosome, and the mar chromosome was considered as r(15) (p11.1q11.1). The second patient had a karyo-type of 47,XX,del(15)(q11.200→q11.207), + idic (15)(pter → q11.1::q11.1→pter). The complex breakage and reunion involving the 15q11.2 regions of the father's homologous chromosomes 15 at meiosis appeared to have resulted in the idic(15) and the del(15) chromosomes. These cytogenetic findings suggest that the PWS chromosome region may be localized on the very proximal portion of band 15q11.2.     

Non-reciprocal translocation (5;15), isodicentric (15) and Prader-Willi syndrome

A non-reciprocal translocation (5;15) and an isodicentric (15) resulting in trisomy 15pter----15q1?3 and monosomy 5qter [46,XY,-5,-15,+der(5)t(5;15) (5pter----5q35::15q13----15qter),+idic(15) (pter----q1?3::q1?3----pter)] was found in a 28-year-old profoundly retarded male resident of a state institution. Early developmental history and childhood and adult physical findings resembled those of Prader-Willi syndrome (PWS) patients. The parents' unbanded chromosomes were normal. Blood groups of parents and propositus were uninformative with regard to identifying gene deletions or duplications.     

Identification,molecular cloning,and characterization of the chromosome 12 breakpoint cluster region of uterine leiomyomas

Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosome 12 aberrations has indicated that their chromosome 12 breakpoints map between linkage locus D12S8 and the CHOP gene. Here, we report fluorescence in situ hybridization (FISH) and molecular cloning studies of the chromosome 12 breakpoints in a panel of seven such uterine leiomyoma cell lines; five with the frequently observed t(12; 14)(q15;q24), one with t(12;15)(q15;q24), and one with ins(12;11)(q14;q21 qter). Directional chromosome walking studies starting from D12S8 and the CHOP gene resulted in the isolation of a relatively large number of overlapping YAC clones, including Y5355 (465 kbp), Y7673 (360 kbp), and Y9899 (275 kbp). In total, the inserts of these three YAC clones span an 800 kbp long and presumably contiguous stretch of human genomic DNA. All chromosome 12 breakpoints of the uterine leiomyoma cell lines studied were found by FISH analysis to be mapping within a 445 kbp subfragment of this region and, furthermore, to be dispersed over a DNA region which is at least 150 kbp in size. The chromosome 12 breakpoint of t(12;14)(q15;q24) in uterine leiomyoma cell line LM-30.1/SV40 was tentatively mapped within the 60 kbp region between YAC clones Y9899 and Y5355. From this 60 kbp region and close to sequencetagged site RM99, we isolated probe pRM 118-A, which showed in Southern blot analysis that it detected a rearrangement in LM-30.1/SV40 DNA, and generated restriction maps of the normal and rearranged genomic DNA regions detected with this probe. Finally, we molecularly cloned part of one of those rearranged DNA fragments using a vectorette-PCR-based technique and demonstrated that it consisted of 12q13-q15 sequences fused to DNA sequences derived from 14q23-24 and most likely represented the translocation junction on der(14) in LM-30.1/SV40 cells. Our studies strongly suggest that we have identified and isolated the uterine leiomyoma cluster region of chromosome 12 breakpoints, which we designate ULCR12, and molecularly cloned and characterized the der(14) translocation junction in cells derived from a uterine leiomyoma carrying the frequently observed t(12;14)(q15;q24).     

Origin of a paternal (13q; 15q) translocation leading to dup(13q) in two half sibs

We report on two half-sibs, a male and a female with dup(13)(q1405 → qter) that resulted from a der(15),t(13;15)(15qter → 15q25::13q1405 → 13qter), h +, pat. Their manifestations were similar to those with duplication of the distal half 13q. The father was a balanced de novo translocation carrier. Since the der(15) had a long secondary constriction, it was possible to trace the site of the mutation to the germ cell of the patients paternal grandmother who had this distinctive long secondary constriction in one of her normal 15 chromosomes.     

Origin of a paternal (13q;15q) translocation leading to dup(13q) in two half sibs

We report on two half-sibs, a male and a female with dup(13)(q1405 leads to qter) that resulted from a der(15),t(13;15)(15qter leads to 15q25::13q1405 leads to 13qter), h+, pat. Their manifestations were similar to those with duplication of the distal half 13q. The father was a balanced de novo translocation carrier. Since the der(15) had a long secondary constriction, it was possible to trace the site of the mutation to the germ cell of the patients paternal grandmother who had this distinctive long secondary constriction in one of her normal 15 chromosomes.     

Genetic counseling in carriers of reciprocal chromosomal translocations involving long arm of chromosome 16

Families with balanced chromosomal changes ascertained by unbalanced progeny, miscarriages, or by chance are interested in their probability for unbalanced offspring and other unfavorable pregnancy outcomes. This is usually done based on the original data published by Stengel-Rutkowski et al. several decades ago. That data set has never been updated. It is particularly true for the subgroup with low number of observations, to which belong reciprocal chromosomal translocations (RCTs) with breakpoint in an interstitial segment of 16q. The 11 pedigrees from original data together with the new 18 pedigrees of RCT carriers at risk of single-segment imbalance detected among 100 pedigrees of RCT carriers with breakpoint position at 16q were used for re-evaluation of the probability estimation for unbalanced offspring at birth and at second trimester of prenatal diagnosis, published in 1988. The new probability rate for unbalanced offspring after 2 : 2 disjunction and adjacent-1 segregation for the total group of pedigrees was 4 +/- 3.9% (1/25). In addition, the probability estimate for unbalanced fetuses at second trimester of prenatal diagnosis was calculated as 2/11, i.e. 18.2 +/- 11.6%. The probability rates for miscarriages and stillbirths/early deaths were about 16 +/- 7.3% (4/25) and <2% (0/25), respectively. Considering different segment lengths of 16q, higher probability rate (0/8, i.e. <6.1%) for maternal RCT carriers at risk of distal 16q segment imbalance (shorter segment) was obtained in comparison with the rate (0/10, i.e. <4.8%) for RCT at risk of proximal segment imbalance (longer segment). It supports findings obtained from the original data for RCT with other chromosomes, where the probability for unbalanced offspring generally increased with decreasing length of the segments involved in RCT. Our results were applied for five new families with RCT involving 16q, namely three at risk of single-segment imbalance [t(8;16)(q24.3;q22)GTG, ish(wcp8+,wcp16+;wcp8-,wcp16+), t(11;16)(q25;q22)GTG, and t(11;16)(q25;q13)GTG] and two with RCT at risk of double-segment imbalance [t(16;19)(q13;q13.3)GTG, isht(16;19)(q13;q13.3) (D16Z3+,16QTEL013-D19S238E+,TEL19pR-; D16Z3-, D19S238E-,TEL19pR+), and t(16;20)(q11.1;q12)GTG, m ish,t(16;20)(wcp16+,wcp20+;wcp16+,wcp20+)]. They have been presented in details to illustrate how the available empiric data could be used in practice for genetic counseling.     

Cytogenetic and molecular studies in patients with chronic myeloid leukemia and variant Philadelphia translocations

Out of 105 Philadelphia (Ph) positive chronic myeloid leukemia patients analyzed, six (5.7%) carried a variant Ph translocation, namely t(6;9;9;10;22)(q24;p13;q34;p15;q11); t(9;13;22)(q34;q21;q11);der(2)(2pter----2q31::9q21---- 9q34::22q11----22qter) and der(9)t(2;9) (9pter----9q21::2q31----2qter);t(7;9;22)(q11;q34 ;q11), 14q + ;t(7;9;22)(q35;q34;q11), and t(9;11;22) (q34;q13;q11), respectively. Five of these patients were analyzed with Southern blotting. Three of them showed an atypical molecular pattern; namely, the patient with t(9;13;22) showed no rearrangement in the breakpoint cluster region (bcr), the patient with t(7;9;22)(q35;q34;q11) showed a 3' deletion, and the patient with t(7;9;22), 14q + showed a bcr rearrangement 3' to the exon 4 of the M-BCR. Chromosome in situ hybridization studies demonstrated that in patient one, a two-step translocation occurred: the first step moved the 3' bcr from chromosome 22 to chromosome 9, and the second moved the terminal part of 22q, carrying the c-sis protooncogene, to 10p. Variant Ph translocations appear to be associated with atypical molecular breakpoints.     

Molecular and cytogenetic studies of the Prader-Willi syndrome.

Twenty-seven subjects with the Prader-Willi syndrome (PWS) were studied. Sixteen (59%) had a cytogenetic deletion involving chromosome 15q11-13. Nine were non-deletional and two patients had structural rearrangements of chromosome 15: 47,XY, + del(15)(pter----q12), var(15)(p11) and 45,XX,t(14q15q). At the DNA level, a greater proportion of patients (74%) showed loss of one chromosome 15q11-13 allele using a combination of densitometry and RFLP analysis. Deletion sizes were variable with 13 of 20 detectable both cytogenetically and with probe pML34 (D15S9). The remaining seven had microdeletions at the pML34 locus. Heterogeneity was further seen in three subjects who had cytogenetic deletions but normal DNA studies. In one patient there was evidence of a duplication at the pML34 locus. A new molecular rearrangement was identified with probe p3.21 (D15S10) in two patients and their mothers. Fifteen family studies were performed. In all 10 families where there was a molecular deletion, this was shown to have arisen de novo. DNA mapping confirmed that the paternal 15q allele was lost in three patients with PWS.     

Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11-13) by YAC cloning and FISH analysis.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.     

A case of Prader – Willi syndrome arising as a result of familial unbalanced translocation t(11;15)(q25;q13)

We report on a case of Prader-Willi syndrome (PWS) with a true reciprocal unbalanced translocation, 45,XX,-15,der(11)t(11;15)pat. The proposita was diagnosed clinically as having severe PWS. Molecular studies revealed loss of the paternal methylation pattern at locus D15S63 and a deletion encompassing the loci from at least D15S10 to D15S97 of paternal chromosome 15. FISH studies confirmed the deletion of 15q11-q13 region and the presence of two telomeres on all chromosomes. The proposita's father, the father's sister and their mother are all carriers of the same balanced translocation t(11;15)(q25;q13). By genomic imprinting we would expect that if the father's sister were to give birth to a child with the same unbalanced translocation as the proband, it would be affected by Angelman syndrome.
To date, a similar familial unbalanced translocation due to loss of the small chromosome 15 derivative has not been described.     

A de novo complex chromosomal rearrangement with a translocation 7;9 and 8q insertion in a male carrier with no infertility

A de novo complex chromosomal rearrangement (CCR) involving chromosomes 7, 8 and 9 in a male carrier was ascertained through his healthy wife's recurrent spontaneous abortions. Six pregnancies over eight years resulted in four spontaneous abortions and two livebirths who died perinatally due to abnormal vital signs. Cytogenetic analyses utilizing high resolution chromosome banding technique showed a deletion of band in a der(7) chromosome and an extra band inserting at 8q21.2. Another extra band was also observed at the band 9p24, but it could not be karyotypically determined. Fluorescent in-situ hybridization using chromosome 7 and 8 specific microdissected library as probes confirmed the insertion of a segment from the translocated chromosome 7 into a chromosome 8, and additionally revealed a translocation between chromosomes 7 and 9. The karyotype of the CCR carrier was determined as 46,XY,t(7;9)(q22;p24),ins(8;7)(q21.2;q22q32).ish der(9)(wcp7+);ins(8;7)(wcp8+,wcp7+). Comparing with previously reported male CCR carriers with our case, we conclude that male CCR carriers may not always present with infertility or subfertility phenotypes. This may suggest that rare transmission of male carriers could result from abnormal chromosomal rearrangements during meiosis and gametogenesis in addition to frequent infertility.     

Common chromosome aberrations in the proximal type of epithelioid sarcoma

A new case of the proximal type of epithelioid sarcoma with a complex karyotype 70-98 <4N>,XX,-X,-X,+5,i(5)(q10),+7,del(7)(q31),i(8)(q10)x3 approximately 4,del(12)(p13),der(18)ins(18:?) (q11;?)del(18)(p11). ish der(18)ins(18;X)del(18)(p11)(wcp18+,wcpX+),+20,+20,dmin [cp9] is described. Both, dual-color FISH using probes specific for OATLI1/OATL2 genes and RT-PCR analysis excluded the presence of t(X;18), typical for synovial sarcoma. Our case together with the previously published ones suggest that the presence of i(8)(q10), losses of 12p and 18p together with the gain of chromosome 20 may represent a common cytogenetic aberrations in the proximal type of epithelioid sarcoma.     

Deletions of proximal 15q and non-classical Prader-Willi syndrome phenotypes

A deletion of the long arm of chromosome 15 (usually involving bands 15q11-q12) has been seen in approximately 50% of Prader-Willi syndrome (PWS) patients [Ledbetter et al, 1982]. However, 14 patients with non-PWS (or atypical PWS) phenotype with 15q deletion indicate great clinical variability. A deletion was found in a propositus with a de novo translocation [45,XY, -15, -22, +rec(15;22) (22pter----22q13.2::15q14----15qter)], who had anomalies not normally observed in PWS patients. Activities of several enzymes mapped to the involved chromosomes were studied in the patient and control individuals. A 50% decrease in the level of arylsulfatase-A confirmed a small deletion in 22q(22q13.2----qter), and additional studies localized more precisely the loci for alpha-mannosidase (cytoplasmic) and beta-galactosidase.     

Atypical 18p- syndrome associated with partial trisomy 16p in a chromosomally unbalanced child of consanguineous parents with an identical balanced translocation

A 2 month old male infant was found to have mild growth retardation, prominent forehead, low set ears, low nasal bridge, rounded facies, cleft palate, webbed neck, shawl scrotum, and absent right kidney. The propositus, a product of a consanguineous marriage, had extremely rare abnormal cytogenetic findings. His karyotype contained three derivative chromosomes that originated from a familial translocation, t(16;18)(p13.3;p11.2) carried by both parents. Based on parental studies, the infant's unbalanced karyotype was defined as: [46,XY,t(16;18)(p13.3;p11.2), der(18)t(16;18).ish t(16;18)(16ptel-,16qtel+,18ptel+,wcp16+,wcp18+;16ptel+,18ptel-,wcp16+,wcp18+), der(18)t(16;18)(16ptel+,18ptel-,wcp16+,wcp18+)]. We describe this child at 2 months of age with a follow up at 4 1/2 years, exhibiting a mixed clinical picture with features of both 18p- and partial trisomy 16p13.3.     

X;14 translocation:an exception to the critical region hypothesis on the human X-chromosome

We report on a family in which an X;14 translocation has been identified. A phenotypically normal female, carrier of an apparently balanced X-autosome translocation t(X;14)(q22;q24.3) in all her cells and a small interstitial deletion of band 15q112 in some of her cells had 2 offspring. She represents a fifth case of balanced X-autosome translocation with the break point inside the postulated critical region of Xq(q13 q26) associated with fertility. The break point in this case is located in Xq22, the same band as in four previously published exceptional cases. In most of her cells, the normal X was inactivated. Her daughter, the proposita, has an unbalanced karyotype 46,X,der(X), t(X;14)(q22;q24.3)mat, del(15)(q11.1q11.3)mat. She is mildly retarded and has some Prader-Willi syndrome manifestations. She has two normal 14 chromosomes, der(X), and deletion 15q11.2. Her clinical abnormalities probably could be attributed to the deletions 15q and Xq rather than 14q duplication. In most of cells, der(X) was inactivated. We assume that spreading of inactivation was extended to the 14q segment on the derivative X. Late replication and gene dose studies support this view. Another daughter, who inherited the balanced X;14 translocation and not deletion 15 chromosome, is phenotypically normal.     

Molecular study of the Prader-Willi syndrome: deletion, RFLP, and phenotype analyses of 50 patients.

Deletion and RFLP studies with 5 cloned DNA markers localized at 15q11.2 were performed in 50 patients with the Prader-Willi syndrome (PWS). A one-copy density (deletion) for at least one of 4 loci, D15S9, D15S11, D15S10, D15S12, was detected in 32 (64%) of the 50 patients; deletions of each of the 4 loci were found in 29, 30, 29, and 28 patients, respectively. Three patients showed 4 or more copy density for D15S12 locus, in addition to deletions. The remaining 18 patients showed two-copy densities for each of the 4 loci. A common site of rearrangements among our 32 patients as well as the reported patients seemed to be confined to a segment between D15S9 and D15S11, suggesting the putative PWS gene locus in this segment. Of 6 patients who have cytologic deletions but did not show any molecular deletions, 3 have normal size of hands and feet, and 4 have normally pigmented skin and hair. The normal pigmentation was also observed in 3 patients who had small molecular deletions in the examined 5-locus segment. These observations may support the conception of contiguous gene syndrome. RFLP analysis demonstrated maternal uniparental isodisomy of chromosomes 15 in both a patient with 45,t(15q;15q) and a karyotypically normal patient. Based on the results of the present study, a new model is proposed to explain the occurrence of PWS with a variety of chromosome abnormalities, including partial monosomy, disomy, trisomy, and/or tetrasomy for 15q11.2. The normal development may require an even or more "number ratio" of paternally derived allele(s) to maternally derived allele(s) of the gene(s) localized at 15q11.2, and a disturbance of the ratio would lead to the PWS phenotype.