Temtamy preaxial brachydactyly syndrome (TPBS; MIM 605282) was first reported in 1998 in an Egyptian patient as a new autosomal recessive multiple congenital anomaly syndrome characterized by bilateral, symmetric preaxial brachydactyly and hyperphalangism of the digits, facial dysmorphism, dental anomalies, sensorineural hearing loss, and delayed motor and mental development (Temtamy et al., 1998). An additional Egyptian patient with similar manifestations apart from hearing loss was further described by Temtamy and Aglan (2008).
The TPBS causative gene studied in the two previously reported Egyptian patients and in an additional three sibs from a consanguineous Turkish family was first reported at the European Society of Human Genetics (ESHG) annual meeting in Vienna, 2009 (Li et al., 2009). Molecular studies were carried out on an additional patient from Sri Lanka and on three Pakistani sibs with TPBS (Race et al., 2010), in addition to those presented at the ESHG meeting (2009) by Li et al. (2010). The researchers showed that loss of chondroitin synthase 1 (CHSY1) function underlies the pathogenesis of autosomal recessive TPBS. CHSY1 encodes an evolutionarily conserved type II transmembrane protein comprising a Fringe motif and a glycosyltransferase domain, whose combined enzymatic activity is crucial for the biosynthesis of chondroitin sulfates. The researchers also obtained similar zebrafish phenotypes after CHSY1 knockdown as well as overexpression, which might explain why in humans, brachydactyly can be caused by mutations leading either to loss or to gain of bone morphogenetic protein/growth and differentiation factor signaling. This was further supported by a back-to-back manuscript by Tian et al. (2010) reporting on two affected siblings with TPBS, offspring of consanguineous Jordanian parents. The researchers detected a truncating frameshift mutation in the same gene CHSY1 and concluded that CHSY1 is a secreted FRINGE enzyme required for the adjustment of NOTCH signaling throughout human and fish embryogenesis and particularly during limb patterning (Tian et al., 2010).
In this study, we contribute to the literature five additional Egyptian TPBS patients with CHYS1 mutations aiming to identify the clinical manifestations in all reported cases in order to define the phenotypic spectrum of this syndrome.
Patients and methods
Five Egyptian patients (four boys, one girl) from three unrelated families were diagnosed at the Limb Malformations and Skeletal Dysplasia Clinic, National Research Centre (NRC), with TPBS. Two families had two similarly affected sibs each. All patients were offspring of consanguineous parents. Their age at presentation ranged from 1 year and 10 months to 7 years and 5 months. Pedigree analysis, clinical and orodental examination, anthropometric measurements, radiological studies of limb anomalies, and bone densitometry (DEXA) were carried out for all cases. Intelligence quotient assessment, hearing test by audiometry, eye examination, abdominal ultrasound, and brain imaging were carried out for selected cases.
Genomic DNA was isolated from whole-blood samples using the standard phenol/chloroform extraction method. To check for CHSY1 mutations, the following PCR primers were used: E1F (5′-GAGCTAAGCCGGAGGATGTG-3′) and E1R (5′-ATCCCGCCTCTGATCTTTTC-3′) for exon 1; E2F (5′-GAACCAAGCAGGCCAAGTAG-3′) and E2R (5′-CCAACTTCAACCCTCAAAGA-3′) for exon 2; E3F1 (5′-TTTGTGTGCTGTGGTCCATT-3′) and E3R1 (5′-GTAAATACGCGTGCCTCCTC-3′), E3F2 (5′-TGGAGATGATCAATGCCAAC-3′) and E3R2 (5′-TGGGTCATACTGGCTGAAGA-3′), and E3F3 (5′-TGCCTGTGTCTGGAGAGTTTT-3′) and E3R3 (5′-AGGCAAACACTGATCACCTTC-3′) for exon 3.
PCR reactions were carried out at a final volume of 50 μl containing 100 ng template DNA using HotStarTaq DNA polymerase (Qiagen, New York, USA). The PCR thermal cycling program was set as follows: 95°C for 15 min, followed by 35 cycles of 45 s denaturation at 95°C, 50 s annealing at 56°C, and 50 s of extension at 72°C, with a final extension at 72°C for 10 min. PCR products were gel recovered using the QIAquick Gel Extraction Kit (Qiagen). Sequence analysis was carried out using the BigDye Terminator cycle sequencing kit (Applied Biosystems, California, USA), and products were run on a 3730 DNA Analyzer (Applied Biosystems).
A 7.5-year-old boy, the first child of apparently healthy parents who are first cousins, was referred to us because of short stature and hand anomalies. He has a normal female sib and a similarly affected younger brother (Fig. 1a). Pregnancy and delivery histories were unremarkable. He had a normal history of motor development. On examination, frontal bossing, a round flat face, wide palpebral fissures, and microstomia and micrognathia were noted (Fig. 1b). Orodental manifestations included a high arched palate and a deep overbite. Skeletal examination indicated brachydactyly with radial deviation of the first to third fingers, clinodactyly of the third and fourth fingers, and camptodactyly of the fourth and fifth fingers, with skin webbing between the fingers (Fig. 1c). Both feet had low inserted broad and short big toes, with clinodactyly of the second to fifth toes and a wide gap between the big toes and the second toes (Fig. 1d). Mild pectus excavatum was noted. Muscle tone and reflexes were normal. No abnormalities in the external genitalia were noted. Radiographs of both the hands showed preaxial brachydactyly with hyperphalangism of the index fingers and flexion deformity in the left second proximal and distal phalanges (Fig. 1e). His bone age was severely delayed. Short toes with hypoplastic medially deviated middle and terminal phalanges were detected on radiographs of both feet. An extra phalanx was seen at the right toes (Fig. 1f). His height was 103.3 cm (−3.9 SD); the weight and head circumference measurements were consistent with his age. A learning disability was reported. Stimulation tests indicated a normal growth hormone level. The thyroid profile was normal. Serum calcium, phosphorus, and alkaline phosphatase were normal. No abnormalities were detected by computed tomographic scan of the brain. Fundus examination was normal. DEXA indicated osteopenia at the femur and the spine (Z-scores: −1.34 and −1.21, respectively). Abdominal ultrasound indicated mild hepatosplenomegaly.
A 4-year-old boy, younger brother of patient 1, was studied. On examination, he was found to be similarly affected as his older brother (patient 1). Facial features and digital anomalies are shown in Fig. 1g–k.
Molecular studies on both the patients indicated a (TGT>CGT; c.2251T>C) homozygous mutation in exon 3 of CHSY1, causing a missense mutation (p.C751R) in the extracellular type-A glycosyltransferase domain of CHSY1 (Figs 4a and 5a, d). Each parent was heterozygous for the same single nucleotide polymorphism.
A 6.5-year-old boy, offspring of parents who are first cousins, was studied. He has a normal older brother and had a twin sib who died at birth. There was a mild delay in motor development. On examination, double hair whorl, frontal bossing, a round flat face, wide palpebral fissures, malar hypoplasia, microstomia, micrognathia, a short neck, and low-set nipples with redundant skin at the abdomen were observed (Fig. 2a). Orodental manifestations included a highly attached thick upper labial frenum, enamel hypoplasia, and hypocalcification in addition to microdontia and a talon cusp in the upper central incisors (Fig. 2b). A panoramic view indicated absent lower permanent first incisors and confirmed talon cusps. Hand examination indicated preaxial brachydactyly with short, low inserted thumbs; a wide space between the first and the second fingers, clinodactyly of the fifth fingers, and abnormal dermatoglyphics (Fig. 2c). The patient had limited supination of both elbows. Knock knees, brachydactyly with bilateral low inserted broad big toes, a wide gap between the first and the second toes, and bilateral medial deviation of all toes more on the left side were observed (Fig. 2d). Exaggerated hyperlordosis with mild scoliosis were additional skeletal features. He had hypotonia, normal reflexes, and normal external genitalia. A radiograph of both the hands showed short first and second metacarpals, an extra phalanx at the index fingers, flexion deformity of the third metacarpophalangeal joints, and hypoplastic pointed distal phalanx of fifth fingers (Fig. 2e). Short broad delta epiphyses of the first metatarsals, short quadrangular proximal phalanx, and hyperphalangism of the first toes were detected on a radiograph of both feet (Fig. 2f). His height at 7.5 years of age was 107 cm (−3.0 SD) and his head circumference was 49.5 cm (−2.0 SD); his weight was the mean weight for his age. Psychological assessment indicated a cooperative child with delayed language, and his intelligence quotient was within the category of mild mental subnormality. The thyroid profile was normal. MRI of the brain indicated a hypovoluminous peritrigonal white matter with an abnormal white matter signal suggestive of postischemic brain insult (Fig. 2g). Fundus examination was normal. Hearing assessment showed bilateral otitis media. DEXA indicated borderline osteoporosis at the femur (Z-score: −2.09) and osteopenia at the spine (Z-score: −1.36). Abdominal ultrasound indicated mild hepatosplenomegaly.
Molecular studies showed a homozygous single nucleotide polymorphism (GGG>TGG; c.664G>T) in exon 2 of CHSY1, leading to a missense mutation, p.G222W, in the FRINGE domain of the CHSY1 enzyme (Figs 4b and 5b, d). Both parents were heterozygous for the same mutation.
A 6.5-year-old boy, offspring of apparently normal parents who are first cousins, was studied. He has a normal female sib and a younger similarly affected sister (Fig. 3a). Pregnancy and delivery histories were unremarkable. He had a normal history of motor development. On examination, frontal bossing, a flat face with malar hypoplasia, wide palpebral fissures, and microstomia and micrognathia were noted (Fig. 3b). Orodental manifestations included a pseudo lower labial cleft, a thick upper labial frenum, a high arched palate, and enamel hypocalcification. Hand examination indicated brachydactyly, low inserted broad thumbs, short second and third fingers with a medial deviation of the left index finger, and broad metaphyses at the wrists (Fig. 3c). Both feet had low inserted broad and short big toes with a wide gap between the big toes and the second toes and a medial deviation of the second to fifth toes (Fig. 3d). Muscle tone and reflexes were normal. No abnormalities in the external genitalia were noted. A radiograph of both the hands showed short first metacarpals with hyperphalangism of the first and second fingers, short proximal phalanges of all fingers, and short middle and terminal phalanges of first to third fingers (Fig. 3e). A radiograph of both feet indicated a bilateral adducted forefoot with replacement of the first metatarsal by short deformed bones, short toes with hypoplastic medially deviated phalanges, and hyperphalangism of the big toes (Fig. 3f). His anthropometric measurements were consistent with his age, although relatively short limbs were noted. Serum calcium, phosphorus, and alkaline phosphatase were normal. No abnormalities were detected by computed tomography scan of the brain. Hearing assessment was normal.
A 2-year-old girl, the younger sister of patient 4, was studied. On examination, she was found to be similarly affected as her older brother. The facial features and digital anomalies are shown in Fig. 3g–k.
Molecular studies on both the patients indicated compound heterozygosity in exon 3, CCT>TCA (c.1075C>T) and AGA>ACA (c.1763G>A), causing, respectively, the heterozygous missense changes p.P359S and p.R588T (Figs 4c and 5c, d).
Table 1 presents the phenotype analysis of our five new reported cases with TPBS and the 11 previously reported cases (all associated with autosomal recessive CHSY1 mutations) in order to define the phenotypic spectrum of the syndrome.
The present study reports on five additional Egyptian patients, offspring of consanguineous parents with the TPBD phenotype, including the characteristic facial features and digital anomalies reported previously by Temtamy et al. (1998). The five patients from three families were found to have CHYS1 missense mutations.
Three of the described TPBS cases in this report had a novel homozygous mutation in either exon 2 or 3 of the CHYS1 gene, leading to missense mutations in residues that are highly conserved across vertebrate and invertebrate species (Fig. 5). Two sibs were compound heterozygous and had a milder phenotype. The occurrence of compound heterozygous Egyptian sibs, offspring of consanguineous parents, suggests a high mutation rate in the community, which warrants further investigations. The mutation rate for CHSYI should be determined in various populations.
Phenotype analysis of the 16 reported cases indicates that preaxial brachydactyly, hyperphalangism, camptodactyly, and clinodactyly are universal findings in the syndrome. Mental subnormality, short stature, a round face, relative microcephaly, a wide-eye look, malar hypoplasia, dental anomalies, microstomia, micrognathia, and osteopenia or osteoporosis were common findings. Follow-up of the first reported case in the literature by the first two authors of this manuscript indicated the progressive course of the syndrome with the development of kyphoscoliosis, pectus excavatum, osteoporosis, and degenerative cortical and cerebellar developments (Temtamy et al., 2010). This emphasizes that follow-up of affected patients is necessary to detect the progressive nature of the syndrome, the appearance of brain changes using MRI, and the deafness apparent with age in some cases.
Of importance to the role of functional polymorphisms of CHSY1 in the general populations is a recent genome-wide association meta-analysis that reported that allelic variants near CHSY1 were significantly associated with central corneal thickness in mixed Asians (Cornes et al., 2012). This finding lends further support to CHSY1’s role during zebrafish and human eye morphogenesis as reported by Tian et al. (2010).
Awareness of the characteristic findings in the syndrome is important for a proper diagnosis and further molecular studies. TPBS is an easily recognizable dysmorphic syndrome. The rarity of worldwide reports could be because of underdiagnosis. Clarkson et al. (2004) described a case as the Catel–Manzke syndrome and questioned the possibility of an extended phenotype or a probable new syndrome. After reviewing the manuscript, the possibility of TPBS in this patient was considered by Temtamy (2005).
Because of some phenotypic overlap with other preaxial brachydactyly syndromes, further clinical and molecular research is required to define a possible genetic heterogeneity, genotype–phenotype correlations, milder expressivity in compound heterozygotes, and the roles of other genes and their encoded proteins in digital differentiation and growth. It is important to integrate CHSY1 function in the cascade of signaling events controlled by the bone morphogenetic protein/growth and differentiation factor pathway, which is most often deregulated in human brachydactylies. In particular, it will be pivotal for understanding the role of CHSY1 in characterizing its proteoglycan targets. These targets should be post-translationally modified by the covalent addition of chondroitin sulfate moieties, and they serve as essential glycosminoglycans during limb patterning and outgrowth.
The authors thank the patients and their families for their cooperation.
Conflicts of interest
There are no conflicts of interest.
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