TWIST1 Gene Variants Cause Craniosynostosis with Limb Abnormalities in Asian Patients

Abstract

The TWIST1 gene codes for a highly conserved transcription factor in a basic helix–loop–helix transcription factors family. The pattern of inheritance is autosomal dominant in Saethre–Chotzen syndrome, Robinow–Sorauf syndrome, and Sweeney–Cox syndrome. Major features of these syndromes include coronal synostosis, vision problems, and deafness, and facial features include hypertelorism, low-set ears, arched eyebrows, beaked nose, maxillary hypoplasia, and other dysmorphisms including broad great toes, clinodactyly, brachydactyly, and cutaneous syndactyly. TWIST1 (bHLH) transcription factor regulates the formation of head and limbs in the embryo. We describe three families affected with craniosynostosis in whom a sporadic TWIST1 variant was identified on whole exome sequencing, chromosomal microarray, and Sanger sequencing.

Introduction

Saethre–Chotzen syndrome (SCS) is a rare autosomal dominant disorder. ^{1 2} It is a craniofacial disorder or craniosynostosis syndrome with limb malformations. ^{3 4 5} The prevalence rate of SCS is approximated as 1/25,000 to 1/50,000 of live births. SCS can be present with or without eyelid anomalies. ^{6 7} It was first described by Haakon Saethre (1931) and Fritz Chotzen (1932). This syndrome is also known as acrocephalosyndactyly (OMIM 601622)

The main manifestation of SCS is craniosynostosis, specifically premature fusion of the coronal sutures which begins within the first 12 months of life. ^{8 9 10} Craniosynostosis, or fusion of cranium bones, affects the facial bones and can lead to facial asymmetry, low set ears, prominent nasal bridge, beaked shape of the nose, eyelid ptosis, and ocular hypertelorism. ^{11 12} Oro-dental manifestations include midface retrusion and lower jaw prognathism. Brachydactyly and cutaneous syndactyly may affect the digits of the upper and lower extremities. ^{13 14} Pathogenic genetic variation for SCS is found on the genomic coordinates (GRCh38): 7:19,113,047-19,117,636 (NCBI), position 7q21 at TWIST1 gene. ^{15 16} TWIST1 gene product is involved in craniofacial and limb development and has basic helix–loop–helix structural domain. Craniosynostosis can also be due to other gene mutations also such as FGFR2 and FGFR3 . ^{17 18} Here, we present three cases of SCS evaluated in the outpatient and inpatient settings and discuss the diverse phenotypic expression of TWIST1 gene mutations.

Methods

The patients were seen in the Genetic Clinic of the Institute, and genetic testing was done with proper pretest and posttest genetic counseling following routine syndrome evaluation and basic radiological and biochemical tests. The patient evaluation and consideration for testing were as per the Declaration of Helsinki. The DNA extraction of the patient samples was done using a Qiagen kit (Qiagen Inc, Netherlands). Sanger sequencing was done using dideoxy nucleotides, and polymerase chain reaction product was separated by capillary electrophoresis and analyzed on Finch TV software using standard established protocols. Next-generation sequencing (NGS) was performed on Illumina platform, and variants were analyzed using set protocols and established bioinformatic tools. A chromosomal microarray was performed using the Affymetrix platform, and HD array was used for identifying the copy number variants.

Results

In the first family, a 3-month-old female was brought to the clinic with complaints of congenital cleft palate and umbilical hernia. She had a birth weight of 2.6 kg and was later found to have neonatal jaundice for which she received phototherapy. Her paternal uncle had an intellectual disability and facial dysmorphism. On examination, she had a length of 62 cm (−0.36Z), weight 5.5 kg (−1.47Z), and occipitofrontal circumference (OFC) 35.5 cm (−4.24Z). She had sparse hair, facial asymmetry, prominent metopic ridge, downward slant of palpebral fissures, low set ears, flat and depressed forehead, hypertelorism, bilateral simian crease, clinodactyly, and cutaneous syndactyly of both hands (involving third and fourth fingers; Fig. 1 ). She had no cardiac murmers but had an umbilical hernia. Renal ultrasound showed insignificant mild left hydronephrosis with an anteroposterior diameter of 3 mm. Thyroid function tests were normal, and echocardiography did not reveal any cardiac defect. Whole exome sequencing was done keeping a possibility of Sweeney Cox syndrome and SCS, which revealed a TWIST1 gene variant in exon 1: NM_000474.4 ( TWIST1) : c.310G > T: p.Glu104*. The coverage of the gene in the NGS assay was 97.83%. The variation in sequence creates a premature translational stop signal p.Glu104* in TWIST1 . The variation of TWIST1 sequence results in a disrupted protein product. This change in the variant is not present in available population databases like ExAC or gnomAD. This premature translational stop signal in sequence has been observed earlier in individuals with SCS (PMID: 9585583). In silico analysis of the variant, different databases such as Varsome, Franklin Genoox, Mutation taster, Mutalyzer, and ClinVar revealed that the variation on TWIST1 is pathogenic as per the American College of Medical Genetics (ACMG) guidelines. The patient carried one copy (heterozygous) of a nonsense variant in TWIST1 gene. This variant is predicted as loss-of-function, and other truncating variants in this gene are also known to cause SCS phenotype.

Fig. 1.

3-year-old patient 1 (P1) with facial dysmorphism ( A ), low set ears and microcephaly ( B ), cutaneous syndactyly ( C ). The paternal uncle of P1 with beaked shaped nose and facial dysmorphism ( D ).

Parents were informed accordingly, and appropriate genetic counseling was provided. The paternal uncle of the patient was also found to have facial dysmorphism, beaked nose, arched eyebrows, and short stature ( Fig. 1D ). Due to this phenotypic appearance similar to SCS in the paternal uncle ( Supplementary Fig. S1 , available in the online version), Sanger sequencing for TWIST1 gene was performed. A heterozygous peak was found at NM_000474.4 ( TWIST1) : c.399G > A: p.Lys133Lys; a synonymous variant is predicted as a variant of unknown significance (VUS) on Franklin Genoox Database ( Fig. 2 ). This variant is novel as it is not reported on clinical population databases such as ClinVar, UniPort, and gnomAD.

Fig. 2.

Sanger sequencing showed heterozygous peak at NM_00047.4( TWIST1 ):399G > A, predicted as variant of unknown significance (VUS) in Franklin Genoox database.

In another family, a 2-year-old male was brought into genetics clinic with brachycephaly low set ears, down slanting eyes, ptosis, small forehead, high arched eyebrows, downward directed mouth angle, and prominent nose ( Fig. 3A ). He had a length of 80.2 cm (−3.67z) height, weight 7.5 kg (−4.94Z), and OFC of 39 cm (−9.23Z). Past medical history included prior admission for respiratory complaints including pneumonia and stranger anxiety. Developmentally, the child walked with support and said bisyllable words. In the setting of severe microcephaly, he was recommended for chromosomal deletion-duplication analysis for SCS. The cytogenomic microarray analysis on the Affymetrix system showed a loss (12.7 Mb) involving chromosome 7 within cytoband region 7p21.3p15.3, indicating monosomy for this region ( Supplementary Fig. S2 , available in the online version). This segment contains 39 OMIM genes including TWIST1 (OMIM 101401). Heterozygous mutation or loss of function in the TWIST1 gene is associated with craniosynostosis-1. Family history was negative for facial asymmetry, abnormal hands feet, and other dysmorphism.

Fig. 3.

2-year-old (P2) with brachycephaly, arched eyebrows, low set ears, down slanting palpebral fissures, and malocclusion of teeth with deletion identified on microarray ( A ); newborn baby with trigonocephaly, facial asymmetry, and thin upper lip who had deletion on chromosome 7 including the TWIST1 gene ( B ).

In the third family, a 6-day-old female, born of a nonconsanguineous marriage, presented with an abnormal head shape. She had trigonocephaly with facial dysmorphism, cleft palate, medially deviated toes, and second to third toe cutaneous syndactyly ( Fig. 3B ). There was no positive family history. Acrocephalosyndactyly syndrome or FGFR2 -related craniosynostosis was suspected. Targeted NGS assay revealed de novo heterozygous 963 kb contiguous deletion encompassing TWIST1 and HDAC9 .

Discussion

SCS is a rare congenital disorder. The main feature in SCS is craniosynostosis. It is an altered cranial development which results in various features including brachycephaly, craniofacial asymmetry, mid-face hypoplasia, prominent eyes, and hypertelorism. ^{1 2 3} The additional malformation includes prominent forehead, ptosis, squint, deviation of nasal septum, beaked nose, low set small ears, and deafness. ^{4 5 6 7} The features in the limbs are brachydactyly with board short thumb, syndactyly, and sometimes radioulnar synostosis. This is associated with intellectual disability which can be mild-to-moderate and short stature is also noted. ^{8 9 10 11 12 13 14 15} Variability of clinical findings in families is observed: one affected member may show abnormal skull shape and fusion of coronal and maybe other sutures, and other individuals may have no clinically evident craniosynostosis. Thus, in milder cases, the phenotype may not be identified. ^{16 17 18 19 20}

The TWIST1 gene provides information in the form of a protein that plays a major role in the developmental phases of an embryo. The protein product of TWIST1 is important in embryonic development for the formation of muscle, bone, and other tissue in the craniofacial regions. ^{21 22 23 24 25 26} It is also important in neural tube development, especially in cranial mesenchyme. TWIST1 has been reported to affect the timing of calvarial suture fusion resulting in craniosynostosis and facial abnormalities. As a transcription factor, the TWIST1 protein is involved in the regulation of various genes like RUNX2 and FGFR2 , which are involved in bone formation. ^{27 28}

FGFR2 expression is mainly detected in the osteogenic fronts that extend into the midsuture of TWIST1 +/− mice, which suggested that the TWIST1 gene normally inhibits FGFR2 expression. In a previous study, the calvarial cells isolated from the SCS patient with TWIST1 variation had decreased FGFR2 levels, which were increased when TWIST1 was normally expressed. The FGFR2 and TWIST1 expression is inversely correlated with each other. The TWIST1 expression inhibits FGFR2 expression. ^{29 30 31 32 33 34}

The TWIST1 downregulation of FGFR2 expression also reduces the RUNX2 and other downstream osteoblast-specific genes in human calvarial osteoblasts. This mechanism was revealed in a study which provides genetic and biochemical evidence for a role of FGFR2 in the altered osteoblast phenotype induced by TWIST1 in SCS patients. ^{18 35 36 37}

We identified a heterozygous known pathogenic variant in the TWIST1 gene causing craniosynostosis in a child in the first family: NM_000474.4 ( TWIST1) : c.310G > T. Interestingly the paternal uncle had some dysmorphism but had a different mutation. The variant NM_000474.4 ( TWIST1) : c.399G > A was found to be a VUS on in-silico analysis, according to ACMG guidelines. ³⁸ SCS, Robinow– Sorauf syndrome, and Sweeney–Cox syndrome have overlapping phenotypes due to heterozygous TWIST1 gene mutations. Sweeney–Cox syndrome is the severe phenotype with significant dysmorphism, cupped low-set ears, and eyelid coloboma; and mutation is in the glutamic acid residue in the important DNA-binding domain of TWIST1 , and mutations reported include p.Glu117Val and p.Glu117Gly. The phenotype in the first child presented here resembles that reported by Miller et al having craniosynostosis, cupped ears, syndactyly, and imperforate anus. ³⁹ TWIST1 and TWIST2 are transcription factors playing significant roles in the development of mesoderm, and pathogenic variants in TWIST2 cause Barber–Say syndrome (BSS). The features of BSS include growth retardation, telecanthus, macrostomia, hirsutism, skin laxity, and in severe form there is ectropion and eyelid agenesis.

In the second affected child in the present report, a large deletion was identified on the chromosomal microarray encompassing chromosome 7p21.1 region that also included the TWIST1 gene. Significant developmental problems are likely to occur in the microdeletion form of SCS. Karyotyping can detect large deletions in 3 to 5% cases of SCS. The third child in the present report, however, showed a small deletion which included the TWIST1 gene and had trigonocephaly. In three Saudi Arabian patients, in a previous study, trigonocephaly was reported with TWIST1 gene variants. ⁴⁰ Recent modifications to include long-read sequencing in NGS and comparison with normal controls have enabled the detection of copy number variants even on NGS data. Some cases of SCS may be due to heterozygous FGFR2 gene mutations. Thus, the presence of facial asymmetry, low frontal hairline, ptosis, beaked nose, hearing loss, and mild hand and feet anomalies including broad great toes and 3-5 syndactyly in a patient with craniosynostosis suggests a diagnosis of SCS. There is a high risk of obstructive sleep apnea, hearing and visual deficits which need appropriate monitoring and management.