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Department of Pediatrics, Keio University School of Medicine (T.O., K.M., G.S.), Tokyo 160-8582, Japan; Department of Radiology, Nasu Chuo Hospital (G.N.), Ohtawara 324-0036, Japan; Department of Orthopedic Surgery, Nagoya University School of Medicine (H.K.), Nagoya 466-8550, Japan; and Department of Pediatric Orthopedics, Center Hospital, Aichi Welfare Center for Persons with Developmental Disabilities (T.H.), Kasugai 480-0392, Japan
Address all correspondence and requests for reprints to: Dr. Tsutomu Ogata, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: . t-ogata{at}po.iijnet.or.jp
Abstract
We report on clinical and molecular findings in a Japanese family consisting of a male infant with SHOX nullizygosity and his four family members with SHOX haploinsufficiency. The male infant had Langer mesomelic dysplasia, the prepubertal sister had idiopathic short stature phenotype with no discernible skeletal features, the father had mild Léri-Weill dyschondrosteosis (LWDC), and the mother and the maternal grandmother had moderate LWDC. The five subjects lacked clinically recognizable short metacarpals, cubitus valgus, high arched palate, short neck, and micrognathia, as well as recurrent otitis media and hearing loss. Fluorescence in situ hybridization and sequence analyses showed that the proband had a pseudoautosomal microdeletion involving SHOX and a C502T missense mutation in the homeobox domain at exon 4, and that the father was heterozygous for the SHOX deletion, and the sister, the mother, and the grandmother were heterozygous for the C502T mutation.
The results, in conjunction with the previous findings, suggest that mesomelic skeletal features such as Langer mesomelic dysplasia and LWDC, which are absent or rare in Turner syndrome, are primarily caused by the SHOX dosage effect and the bone maturing effect of gonadal estrogens, whereas other skeletal features such as short metacarpals, cubitus valgus, and various craniofacial and cervical skeletal stigmata, which are common in Turner syndrome, are largely contributed by a compressive effect of distended lymphatics and lymphedema on the developing skeletal tissues.
SHOX (SHORT STATURE homeobox containing gene) cloned form the approximately 2.6-Mb short arm pseudoautosomal region of the human sex chromosomes is the first gene that has been shown to be relevant to the development of specific features in Turner syndrome (1). Clinical studies in patients with intragenic SHOX mutations and pseudoautosomal microdeletions involving SHOX as the sole disease gene have shown that SHOX haploinsufficiency can cause not only short stature but also Turner skeletal features such as high arched palate, short neck, short metacarpals, cubitus valgus, and Madelung deformity characteristic of Léri-Weill dyschondrosteosis (LWDC) (1, 2, 3, 4, 5, 6). Consistent with the dominant clinical effects and the distribution of skeletal lesions, it has been shown that SHOX escapes X-inactivation (1) and is exclusively expressed in the first and second pharyngeal arches and in the developing distal limb bones of human embryos (5).
Nullizygosity for SHOX has been shown to result in Langer mesomelic dysplasia (LMD) characterized by marked shortening and underdevelopment of long bones in the forearms and shanks. Belin et al. (2) reported a 24-wk fetus with LMD and a pseudoautosomal microdeletion encompassing SHOX occurring in a 45,X karyotype; Shears et al. (3) described a 20-wk fetus with LMD and homozygous pseudoautosomal microdeletions involving SHOX; and Robertson et al. (7) documented a 12-yr-old boy with LMD and homozygous pseudoautosomal microdeletions involving SHOX. These findings provide compelling evidence for the previous hypothesis that LMD is the homozygous expression of the mutant gene for LWDC (8).
However, the prevalent skeletal features relevant to SHOX abnormalities appear to be different between patients with a normal karyotype (1, 2, 3, 4, 5, 6, 7, 8) and those with Turner syndrome (9). Here, we report on clinical findings in a male infant with SHOX nullizygosity and his four family members with SHOX haploinsufficiency and discuss the primary factors involved in the development of skeletal features common in Turner syndrome and those absent or rare in Turner syndrome.
Materials and Methods
Clinical reports
The pedigree of this Japanese family is shown in Fig. 1
. The proband was a male infant born to nonconsanguineous parents at 39 wk of gestation, with a prenatal diagnosis of LMD on fetal ultrasound examinations. He was delivered in a normal cephalic position without asphyxia. At birth, his length was 43.0 cm (-3.7 SD), weight 3.25 kg (+0.1 SD), and head circumference 34.0 cm (+0.4 SD). Subsequently, his developmental milestones were normal. There was no episode of otitis media. At 19 months of age, he remained markedly short, with severe mesomelia and moderate rhizomelia. His auxological data are shown in Fig. 1, with SD scores assessed by the age-matched Japanese references (10, 11 , and the national survey of the Ministry of Education, Science, Sports, and Culture) [no references are available for sitting height (SH), leg length (LL), or SH/LL ratio below 6 yr old or for arm span in all ages]. He exhibited severe short stature but had normal weight and head circumference. Both hands showed ulnar deviation, and there was no clinically recognizable short metacarpal or cubitus valgus. His facial appearance was normal with no discernible high arched palate, short neck, or hypoplastic mandible. He could respond well to small sounds and voices, indicating normal hearing acuity. Roentgenograms showed LMD features such as severely shortened mesomelic bones, markedly curved radii, and hypoplastic distal ulnas and proximal fibulas (Fig. 2). The angle formed between the humerus and the ulna, which was used as an indication of cubitus valgus, was apparently normal.
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). The sister, the father, and the maternal grandmother had short stature, although the mother had low-normal height. The LL was more severely affected than the SH, with resultant increase in the SH/LL ratio, especially in the mother and the grandmother. They exhibited mild to moderate mesomelic appearance, but had no short metacarpal, cubitus valgus, high arched palate, short neck, or hypoplastic mandible. Roentgenograms of the forearms revealed apparently normal findings in the sister and LWDC features in the three adult subjects, including bilateral mild radial curvature in the father, and bilateral moderate radial curvature, decreased carpal angle, and distal radial angulation in the mother and the grandmother. In addition, roentgenograms of the shanks delineated bilateral fibular exostosis in the father and the mother, and left tibial exostosis in the grandmother. The four subjects had no history of recurrent otitis media or hearing difficulty in their daily life, although the father and the mother had a single episode of otitis media. They had normal mental development. Allegedly, two of the six paternal sibs and the paternal father had short stature. Conventional and molecular cytogenetic studies
After obtaining informed consent, chromosome analysis was performed on 50 peripheral lymphocytes of the five subjects by G-banding. Fluorescence in situ hybridization (FISH) analysis was performed for lymphocyte metaphase spreads, using cosmid probes for the Xp/Yp telomeric region at approximately 20 kb from the chromosome end (12), SHOX at the position approximately 500 kb from the Xp/Yp telomere (4), and MIC2 at the position approximately 2.5 Mb from the Xp/Yp telomere (kindly provided by Mitsubishi Kagaku Bioclinical Laboratories, Inc., Tokyo, Japan), together with a cosmid probe for the Xq/Yq telomeric region (12) used as an internal signal control. The probe for the Xq/Yq telomeric region was labeled with biotin and detected by avidin conjugated to fluorescein isothiocyanate, and the remaining probes were labeled with digoxigenin and detected by rhodamine antidigoxigenin.
Mutational analysis of SHOX
Leukocyte genomic DNA of the five subjects was amplified by PCR for each exon of the SHOX gene and its exon-intron boundaries. The primer sequences and the PCR conditions were as described previously (3). Subsequently, the PCR products were purified and subjected to direct sequencing from both directions on an ABI PRISM 310 autosequencer (PE Applied Biosystems, Foster City, CA). For controls, leukocyte genomic DNA from 120 normal Japanese subjects were used with permission.
Results
Conventional and molecular cytogenetic studies
The karyotype was normal in all of the five subjects examined. FISH analysis showed loss of SHOX from the Y chromosome of the proband and from the X chromosome of the father, demonstrating heterozygous SHOX deletion in the two males (Fig. 3). The Xp/Yp telomeric region and MIC2 were present in two copies in the proband and the father, indicating a pseudoautosomal interstitial microdeletion in the two males. The sister, the mother, and the grandmother had two copies of SHOX as well as the Xp/Yp telomeric region and MIC2.
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Direct sequencing revealed a C
T transition at the nucleotide position 502 in the exon 4 of the proband hemizygous for SHOX (Fig. 4A). This C502T transition resulted in a substitution of the 168th arginine codon by tryptophan codon (R168W). This missense mutation was shown to be present in a heterozygous status in the sister, the mother, and the grandmother, and was absent from the father by direct sequencing. The C502T mutation should destroy an MspI restriction site, and this was confirmed by MspI digestion for the PCR products encompassing the exon 4 (Fig. 4B). Furthermore, the C502T transition was confirmed to be absent from 20 control subjects by the direct sequencing analysis and from another 100 control subjects by the MspI digestion analysis.
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The present study showed a heterozygous SHOX deletion in the proband and the father, a hemizygous C502T transition in the proband, and a heterozygous C502T transition in the sister, the mother, and the grandmother (Fig. 1). The SHOX deletion is considered to be transmitted from the paternal X chromosome to the proband’s Y chromosome by a pseudoautosomal recombination during paternal meiosis. The C502T transition is regarded as a true mutation rather than a polymorphism, because it was absent from a total of 240 alleles in the 120 control subjects. Because the C502T transition resides in the homeobox domain (1), it would inhibit a DNA binding capacity of SHOX. Thus, the results indicate SHOX nullizygosity in the proband and SHOX haploinsufficiency in the four family members.
Clinical features in this family are primarily consistent with the previous findings (1, 2, 3, 4, 5, 6, 7, 8). The proband with SHOX nullizugosity had LMD, and the four subjects with SHOX haploinsufficiency had short stature or LWDC. Furthermore, clinical findings of the four subjects are consistent with the notion that gonadal estrogens exert a maturational effect on skeletal tissues that are susceptible to unbalanced premature growth plate fusion because of SHOX haploinsufficiency, facilitating the development of limb skeletal lesions and mesomelic short stature in a female-dominant and pubertal tempo-influenced fashion (4). Although the mother had low-normal height in the presence of moderate LWDC, this would be explained by assuming that she had a high original growth potential, as suggested by her tall SH (Fig. 1). Thus, it is inferred that skeletal features in the mesomelic (and probably also rhizomelic) segments such as LMD and LWDC, which are absent or rare (
7.5%) in Turner syndrome (9), are primarily caused by the SHOX gene dosage and subject to the bone maturing effect of gonadal estrogens.
However, the family members including the proband with SHOX nullizygosity lacked common Turner skeletal features with the prevalence of 35–70%, such as short metacarpals, cubitus valgus, high arched palate, short neck, and micrognathia, as well as recurrent otitis media and hearing difficulty, which could be related to disturbed growth of the cranial base (9, 13). Consistent with this, although hypoplastic mandible has often been described in patients with LWD, other common Turner skeletal features have not been documented in such patients (2, 3, 7, 8, 14). Similarly, although patients with SHOX haploinsufficiency and normal gonadal function often have short metacarpals and cubitus valgus and occasionally exhibit craniofacial and cervical skeletal features, the prevalence of common Turner skeletal features is lower than that of LWDC in such patients (1, 2, 3, 4, 5, 6). These findings imply that some factor(s) other than gonadal estrogens plays a pivotal role in the development of common Turner skeletal features.
In this regard, lymphatic hypoplasia is noteworthy (15). It has been postulated that a lymphogenic gene escaping X-inactivation resides in an approximately 9-Mb region between DMD and MAOA on Xp (16) and in an approximately 4-Mb region between PABY and DYS255 on Yp (17), and that haploinsufficiency of the lymphogenic gene leads to lymphatic hypoplasia in Turner syndrome (18). Lymphatic hypoplasia should cause lymph fluid stasis, resulting in distension of the main and tributary lymphatic ducts and in lymphedema. Thus, it has been suggested that distended cervical lymphatics (cystic hygroma) and facial lymphedema lead to faciocervical anomalies such as webbed neck and malrotated auricle, and that peripheral distended lymphatics and lymphedema results in acral anomalies such as puffy hands and feet and redundant skin (18). It has also been suggested that cystic hygroma and distended para-aortic lymphatics compress the aortic arch, leading to cardiovascular anomalies such as aortic coarctaton, and that distended retroabdominal and iliac lymphatics inhibit normal upward migration and rotation of the kidney, leading to renal malformations such as horseshoe kidney (18, 19). Thus, characteristic soft tissue and visceral anomalies have been regarded as a malformation sequence initiated by lymphatic hypoplasia (18).
This idea of the malformation sequence could also be applied to skeletal lesions. It is possible that cystic hygroma and facial lymphedema exert a compressive effect on the developing skeletal tissues, facilitating the occurrence of various craniofacial and cervical Turner skeletal features. It is also possible that peripheral distended lymphatics and lymphedema exert a mechanical force on the developing skeletal tissues, contributing to the occurrence of short metacarpals and cubitus valgus. In support of this notion, Ogata et al. (16) have reported that the prevalence of common Turner skeletal features tends to be higher in females with large Xp deletions missing the critical region for the lymphogenic gene than in those with small Xp deletions preserving that region, with a statistical significance for short neck, on the basis of genotype-phenotype correlations in 47 patients with various types of Xp deletions. Furthermore, the idea of the malformation sequence would explain why Noonan syndrome is associated with various Turner-like soft tissue and skeletal features, because Noonan syndrome is accompanied by peripheral lymphatic hypoplasia or dysplasia, together with pulmonary lymphangiectasis compatible with right-sided cardiac anomalies such as pulmonary stenosis (20).
In summary, we propose that, of SHOX-related skeletal lesions, LMD and LWDC, which are rare or absent in Turner syndrome, are primarily caused by the SHOX dosage effect and the bone-maturing effect of gonadal estrogens, whereas other common Turner skeletal features are largely contributed by a compressive effect of distended lymphatics and lymphedema on the developing skeletal tissues. Further careful clinical studies will permit a better clarification of the factors involved in the development of skeletal features.
Acknowledgments
Footnotes
This work was supported by a grant for Pediatric Research from the Ministry of Health and Welfare, by Pharmacia Fund for Growth and Development Research, and by a grant from the Foundation for Growth Science.
Abbreviations: FISH, Fluorescence in situ hybridization; LL, leg length; LMD, Langer mesomelic dysplasia; LWDC, Léri-Weill dyschondrosteosis; SH, sitting height.
Received September 25, 2001.
Accepted December 12, 2001.
References
This article has been cited by other articles:
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  M. R. Perkiomaki and L. Alvesalo Palatine ridges and tongue position in Turner syndrome subjects Eur J Orthod, April 1, 2008; 30(2): 163 - 168. [Abstract] [Full Text] [PDF]
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