A Recurrent c.416C>T Variant in the B3GAT3 Gene in the Turkish Population: Report of Two Siblings and Expanding the Clinical Spectrum Free

Linkeropathies are a group of rare connective tissue disorders caused by defects in the enzymes involved in proteoglycan synthesis [1, 2]. Proteoglycans are major components of the extracellular matrix, particularly in cartilage, bone, and cardiovascular systems, and play important roles in cell proliferation, differentiation, development, and cell-cell and cell-matrix interactions [3, 4]. Proteoglycans are structurally complex biomacromolecules and consist of a core protein and attached glycosaminoglycan side chains via the tetrasaccharide linker region [5, 6]. Five enzymes including xylosyl-transferase I and II (encoded by XYLT1 and XYLT2), galactosyltransferase I and II (encoded by B4GALT7 and B3GALT6), and glucuronyltransferase I (encoded by B3GAT3) are responsible for the synthesis of common linker region, and defects in these five enzymes are referred to as linkeropathies [1, 7].

Multiple joint dislocations, short stature, and craniofacial dysmorphism with or without congenital heart defects (MIM # 245600), also known as B3GAT3-related linkeropathy syndrome, is a rare autosomal recessive disorder characterized by mild dysmorphism, joint laxity, contractures, cardiovascular defects, and skeletal abnormalities including osteopenia, pectus deformities, kyphoscoliosis, radio-ulnar fusion, arachnodactyly, spatulate fingers, and feet deformities [7‒9]. According to the nosology of genetic skeletal disorders, 2023 revision, it takes place in group 5, ”Dysplasias with multiple joint dislocations” [10].

Up-to-date, only 34 patients from 20 families have been reported with this rare syndrome [7, 9, 11]. In this study, we report on two siblings with homozygous B3GAT3 variant and expand the clinical spectrum of the B3GAT3-related linkeropathy syndrome.

A 14-year-old boy patient was referred to the pediatric genetic department due to short stature, kyphoscoliosis, and pectus deformity. He was the first child of nonconsanguineous Turkish parents. The family history was unremarkable for any genetic disorders. They had two aborted histories. The pregnancy was complicated with preeclampsia and oligohydramnios. He was born at 36th gestational weeks with a birth weight of 1,800 g (−2.8 SD). Birth length was not available. Bilateral pes equinovarus deformity was noticed at birth, and foot plastering was done. At 3 months of age, limitation of the elbows was detected. Scoliosis was noticed at 3 years old. Neuromotor development milestones were appropriate for age, and he had normal intelligence.

He was evaluated at 14 years old. He did not experience any fracture, joint dislocations, easy bruising, undescended testis, or umbilical or inguinal hernia, but he had delayed and poor wound healing. Audiologic and ophthalmologic evaluations were also normal. Abdomen ultrasound showed splenomegaly at upper limit liver size (spleen: 120 mm in length, 90th centile for age: 115 mm; right liver lobe length: 146 cm, 90th centile for age: 149 mm), rotation abnormality of the left kidney, and increased kidney sizes (left kidney: 120 × 35 mm, 90th centile: 114 mm, right kidney: 114 × 49 mm, 90th centile: 112 mm) [12]. The routine tests for splenomegaly including the complete blood count, peripheral blood smear, and liver function tests were normal, and viral markers were negative. Echocardiography revealed aortic root dilatation. The absence of lower premolar teeth was noted at the orthopantomogram, shown in Figure 1.

On physical examination, long face, blue sclera, tubular nose, bifid uvula, low-set ears, visible veins on the chest, severe kyphoscoliosis, pectus carinatum, limited elbow extension, mild joint laxity, long and spatulate-shaped fingers, genu valgum, hindfoot deformity, hallux valgus, medially deviated 2–5th toes, pes planus, and deep vertical plantar creases were noted (Fig. 1a). His height was 120 cm (−6.0 SD), arm span was 123 cm (suboptimal due to limited elbow motion), weight was 21 kg (−5.2 SD), and head circumference was 46 cm (−6.6 SD).

Radiographs revealed severe kyphoscoliosis (T5-T12 Cobb angle: 120°, T10-L2 Cobb angle: 79°), short and broad ilia, short and wide femoral necks, radio-ulnar synostosis, diaphyseal constriction of metacarpals and metatarsals, broad first metacarpal on the left hand, and hallux valgus deformity (Fig. 2a). With the findings of severe kyphoscoliosis, blue sclera, bifid uvula, radio-ulnar synostosis, foot deformities, and aortic root dilatation, the clinical diagnosis of B3GAT3-related linkeropathy syndrome was considered.

Patient 2, the sibling of Patient 1, was 6 years old. She was born at 39th gestational week with a birth weight of 2,400 g (−2.2 SD). The pregnancy was complicated with oligohydramnios. At birth, pes equinovarus was detected bilaterally, as in Patient 1. Limitation of elbow extension was noticed when she was 3 months old.

She had no history of fracture or dislocation. Neuromotor development, audiologic and ophthalmologic evaluation, and abdominal ultrasound were normal. Echocardiography revealed mild mitral insufficiency. During the initial examination, the patient presented with facial asymmetry that had been ongoing for 2 weeks, and she was undergoing steroid treatment. A cranial CT scan was normal. Upon a second visit, approximately 2 months later, the patient appeared completely normal.

Her height was 98 cm (−4.1 SD), arm span was 94 cm, weight was 16.2 kg (−2.0 SD), and head circumference was 49.5 cm (−1.2 SD). She had decreased periorbital adipose tissue, down-slanting palpebral fissures, mild eversion of lower eyelids, blue sclera, microstomia, low-set and posteriorly rotated dysplastic ears, single palmar crease, increased lumbar lordosis, genu recurvatum, hindfoot deformity, pes planus, and hallux valgus deformity, shown in Figure 1b. Radiographies revealed osteopenia, mild scoliosis, increased lumbar lordosis, radial bowing and radial head dislocation at proximal sides bilaterally, the fusion of lunate and triquetrum bones, and hallux valgus deformity, shown in Figure 2b.

B3GAT3-related linkeropathy syndrome was considered, and clinical-exome sequencing (CES) analysis was performed on Patient 2 using the DNA obtained from peripheral blood leukocytes. The sequencing library was generated using the Clinical Exome Solution v3 capture kit (SOPHiA Genetics SA, Switzerland), and sequencing itself was conducted on the MiSeq system (Illumina Inc., CA, USA). The CES analysis revealed homozygous c.416C>T (p.Thr139Met) (RefSeq: NM_012200.4, Ensembl: ENST0000265471) variant in the B3GAT3 gene.

The identified variant was verified through pool-sequencing in Patient 2, and Patient 1 also had the same homozygous variant. Upon conducting segregation analysis, it was determined that the father, mother, and two additional healthy siblings all carried the variant in a heterozygous state.

The variant was classified as “VUS” according to the ACMG criteria and had a very low frequency in the GnomAD Genomes database (f: 0000131). The CADD score was 27.2 and the MetaRNN score was 0.7925 (pathogenic supporting). It was predicted to be “probably damaging” according to PolyPhen2 (score: 1.000).

B3GAT3-related linkeropathy syndrome is a rare genetic disorder that was first described in 2013 [13]. Since then, a total of 34 patients have been reported, exhibiting a wide range of clinical features [7, 9, 11]. The clinical and radiological characteristics of all patients with the B3GAT3 variants were summarized in Table 1.

The syndrome can present with mild to severe, and even lethal, symptoms. The oldest reported patient was 23 years old, while 8 patients died before the age of one [8, 14, 15]. Craniofacial features include prominent eyes, down-slanting palpebral fissures, blue sclera, midface hypoplasia, microstomia, and low-set dysplastic ears. Bifid uvula/cleft palate is a rare finding; it was observed in Patient 1 [6, 8].

Skeletal findings are joint contractures, joint laxity and dislocations, radio-ulnar synostosis, short limbs, pectus carinatum, kyphosis, scoliosis, spatulate distal phalanges, and foot deformities. Most of the patients (17/25 patients) had variable degrees of short stature, and the current two siblings also had short stature. Radio-ulnar synostosis is a rare condition, and it is an important clinical finding, particularly in the diagnosis of certain syndromes. In the 34 previously reported patients, it is worth noting that 12 of them exhibited radio-ulnar synostosis [9]. Additionally, craniosynostosis was reported in 3 patients, and the presence of radio-ulnar synostosis alongside craniosynostosis led to the suspicion of Antley-Bixler syndrome [15]. It is important to consider Antley-Bixler syndrome in the differential diagnosis of patients presenting with radio-ulnar synostosis and craniosynostosis [15]. Joint dislocation was observed in 26 out of 31 patients, and kyphosis/scoliosis were detected in 13 out of 25 patients [7, 9, 17]. In our study, Patient 1 presented with a severe degree of kyphoscoliosis. The simultaneous presence of developing osteoporosis alongside severe kyphoscoliosis significantly complicates the treatment process. Consequently, in cases of pronounced kyphoscoliosis, a systematic surgical strategy can be devised, involving a gradual reduction of deformity through the implementation of halo gravity traction, ultimately leading to the final surgical intervention. For this specific patient, a deliberate and controlled procedure was executed to achieve surgical correction. In the initial phase, halo gravity traction was administered to the patient under local anesthesia. Traction was started on the same day with a weight of 4 kg. Traction weights were progressively increased, reaching 50% of the patient’s body weight by the eighth week. Following this, the second phase involved posterior surgical instrumentation and fusion. In the third stage, a separate session was dedicated to posterior vertebral corpectomy and cage placement, culminating in the attainment of the final correction. During the second and third stages of the surgical procedures, no impairment was observed in neuro-monitor responses. No complications were noted either intraoperatively or postoperatively. Pre-operative and post-operative images and radiographies are shown in Figure 3.

Recurrent fractures due to osteopenia represent a significant and concerning complication in patients with this condition. It has been documented that fractures have occurred in 9 patients, including neonates, indicating the severity of the issue [9, 15]. In a study by Jones et al. [3], a patient was found to have a history of approximately 25 fractures by the age of 1 year, despite receiving pamidronate treatment. This highlights the challenges in managing fractures in individuals with osteopenia, even with medical intervention.

Cardiovascular defects, such as aortic root or ascending aorta dilatation, atrial septal defect, ventricular septal defect, patent ductus arteriosus, pulmonary stenosis, bicuspid aortic valve, and valve insufficiencies, are frequently observed in B3GAT3-related linkeropathy syndrome, with approximately half of the patients affected [9, 11]. Aortic root dilatation was identified in 6 patients, and Patient 1 exhibited aortic root dilatation as well. He was referred us due to pectus carinatum, scoliosis, and aortic root dilatation, with a suspicion of Marfan syndrome. Similarly, Li et al. [9] described a 2-month-old boy with B3GAT3 variants initially suspected to have neonatal Marfan syndrome. It is important to consider other connective tissue disorders presenting with skeletal deformities and aortic root dilatation, such as Marfan syndrome, Loeys-Dietz syndrome, Beals syndrome, and Shprintzen-Goldberg syndrome, in the differential diagnosis.

Skin involvement in B3GAT3-related linkeropathy syndrome is not commonly observed. While hyperextensible skin and excessive wrinkling on the palms and hands may be present, it is not a typical feature of the syndrome [4, 8]. Additionally, easy bruising and atrophic scarring have only been reported in one patient, as documented [6].

In this report, we have outlined new features associated with this syndrome. These include the absence of premolar teeth, splenomegaly, and carpal bone fusion. Upon investigation, we were unable to identify any underlying cause for the splenomegaly, highlighting the need for further research and reports in this area.

The syndrome is known to be caused by homozygous variants in the B3GAT3 gene, but Bloor et al. [16] reported a patient exhibited clinical findings consistent with the disorder despite carrying a heterozygous c.888 + 262T>G variant. The variant was classified as pathogenic due to its detrimental effect on mRNA splicing efficiency, and they suggested that the truncated protein could potentially exert a dominant negative effect. However, segregation analysis was absent.

Byrne et al. reported the cases of 2 patients with homozygous B3GAT3 variants, both of whom were diagnosed with pseudodiastrophic dysplasia, in which genetic etiology is unknown. One patient, at 16 weeks of gestation, was an abortus, while the other died at 9 months of age. Functional assays revealed a near-complete or complete loss of glucuronyltransferase activity in vitro. This led the authors to propose that the severe and lethal phenotype observed in these patients may be attributed to a complete loss of function resulting from the B3GAT3 variants. Additionally, they noted that the clinical spectrum of B3GAT3-associated disease is diverse and could be linked to varying levels of residual enzyme activity stemming from specific mutations [14].

Hitherto, 17 variants in B3GAT3 have been reported in 34 patients in the literature [7, 9, 11, 18]. The current variant, c.416C>T (p.Thr139Met), has been reported twice before in patients with the origins of Turkey and Tunisia [8, 11]. Although the variant is classified as VUS according to the ACMG criteria, segregation analysis revealed that four unaffected family members were in a heterozygous state, providing strong evidence for its pathogenicity. Regrettably, due to limitations, functional studies could not be conducted to further elucidate the implications of this variant. Considering the lack of consanguinity between the parents in our report, the c.416C>T variant, which was found in 2 of the 4 Turkish families including our family, was assumed to be a frequent variant in the Turkish population.

In conclusion, B3GAT3-related linkeropathy syndrome is a rare genetic disorder with a wide spectrum of clinical manifestations and requires thorough evaluation for cardiovascular, ophthalmologic, and skeletal complications in affected patients. By increasing awareness and expanding the phenotypic spectrum, we aim to improve understanding of this rare syndrome. Early recognition and genetic testing are essential for accurate diagnosis and appropriate management. However, further research and long-term follow-up studies are necessary to gain a comprehensive understanding of the natural history and develop optimal treatment strategies for this rare genetic condition.

We thank the family for their collaboration.

Written informed consent was obtained from the parents for the publication of this case report and accompanying images. Ethical approval was not required since it was a routine diagnostic activity. Ethical approval was not required for this study in accordance with local/national guidelines.

The authors declare no conflict of interest.

Tuğba Daşar: conceptualization, writing, and analysis. Abdulkerim Kolkıran: writing – review and editing and final approval of the version to be published. Abdullah Sezer: analysis, writing – review and editing, and final approval of the version to be published. Ercan Bal and Esra Kılıç: writing – review and editing, supervision, and final approval of the version to be published.

The data are available on request.