Novel Mutation Detection in Craniosynostosis Promotes Characterization, Identification, Gene Expression, Tissue Tengineering and Helps Clinical Practice and Translational Research
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.284349
Source of Support: None, Conflict of Interest: None
Keywords: Craniosynostosis, fibroblast growth factor receptor, genotypic, phenotypic, molecular targeted therapy, Molecular Medicine
Craniosynostosis (CS) syndrome is an autosomal dominant (AD) condition (ADC), classically combines CS and nonsyndromic CS (NSCS) within digital anomalies. The hands and feet have the majority of cases fused due to heterozygous mutations (HMs). The third immunoglobulin-like domain (IgIII) of fibroblast growth factor receptor (FGFR) 2 mutations and this region of the protein were responsible for such kind of syndrome. CS has common craniofacial disorders (CDs) encountered in clinical genetics practice. The overall incidence is 1:2,500 globally (Indian scenario is 1:1,000) lives births. Thirty-five and 75% of SYCSs are caused by hotspots mutations in the FGFR genes. In an other way the TWIST1 gene, at the difference in detection rates (DRs), in different study populations, within CDs. Australia and New Zealand cohort of 630 individuals with a diagnosis of CS. Data were obtained by the Sanger sequencing (FGFR1, FGFR2, and FGFR3 hotspot exons) only. Even the TWIST1 gene had copy number detection higher in TWIST1 gene. Researcher finds out 630 probands and 231 had 80 distinct patterns of mutations. A total of (36%) 80 mutations, 17 novel sequence variants were detected and screened. FGFR1-3/TWIST1-associated syndromes were observed as predictive for mutation detection (MT) and had a statistically significant association between splice site mutations (SSMs). FGFR2 mutation clinically diagnosed with Apert's, Crouzon's, metopic, and Pfeiffer's syndromes had severe clinical phenotypes and was associated with the FGFR2 exon 10 versus exon 8 mutations. The surgical procedures in the presence of a pathogenic mutation had a special target and our interest. As hot spot gene areas were a useful strategy to maximize the success of molecular diagnosis for individuals with CS conditions.
Prospective cohort analysis of clinical records of patients with registered in CS clinic from January 2007 to 2018 was performed.
Diagnosed cases in SYCS and NSCS patients between 3 months and 14 years of age either preoperative or postoperative were included in the study of both groups (SYCS and NSCS).
Patients with primary microcephaly (secondary CS), postural plagiocephaly, incomplete data, no visual perception, and lost to follow-up and those who were not interested to participate the study were excluded from the study.
Diagnostic investigations included with clinical examination and plain X-ray skull (anterior–posterior, lateral, and Towne's view) and noncontrast computed tomography with three-dimensional reconstruction if required. Of 1,080 registered cases, 1,075 cases were satisfying the inclusion and were taken for the study of both the conditions (CS + NSCS).
Blood sample (5 mL) was taken from both the parents along with the child in ethylene-diamine-tetra-acetic acid (vial). For control, 500 healthy children of comparable age group, belonging to the same geographical region were included in this study. Genomic DNA was extracted from peripheral blood lymphocytes by phenol–chloroform extraction method. Primers were diagnosed with FGFR1, FGFR2, FGFR3, FGFR4, TWIST, and MSX mutations in this study were custom-synthesized primers for FGFR1 and FGFR2 genes (Sigma-Aldrich Chemicals Pvt. Ltd., Bengaluru, India).
Polymerase chain reaction
The polymerase chain reaction (PCR) for each sample was performed in 0.2 mL, thin-walled tubes by used 20 ng of DNA. The 2–5 pmol of each primer, 200 mm dinucleotide triphosphates, 10 × PCR buffer, 1.5 mm MgCl2, and 0.5 units of DyNAzyme II DNA Polymerase (Thermo Scientific) were used. The PCR reaction was carried out in a T-100 DNA Engine (Bio-Rad, Hercules, CA, United States). The thermal cyclers under the following conditions: 95°C for 3 min, 35 cycles at 95°C for 30 s, annealing temperature as in for 30 s and 72°C for 1 min/kb, and a final extension at 75°C for 7 min. The amplicon size was verified by gel electrophoresis by running the PCR products on 2% agarose gel with the 100 bp maker (ladder). After the successful amplification, PCR products were digested as per the manufacturer's instructions with the respective restriction endonucleases mentioned in and analyzed through an ethidium bromide-stained 2.0% agarose gel with 50 bp ladder. Finally, PCR purified products were sequenced through the Sangers dideoxy method.
Five hundred fifty children attending the pediatric surgery outpatient department, and without any known birth defects served as “controls.” After taking consent, parents of study subjects were given a personal and telephone interview to obtain information regarding parental and proband medical history, pregnancy and family history, and demographics. Parents were examined clinically in syndromic and nonsyndromic cases complaining with SYCS and NSCS.
Ethical clearance was obtained from the Institute's Ethical Committee.
Statistical analysis was performed by using the SPSS for Windows, version 14.0 (SPSS, Chicago, IL, United States). Chi-square (χ2) test was applied for the assessment of association in two-dimensional contingency tables. Odds ratios and 95% confidence intervals were calculated to measure the relationship between a potential risk factor of cases/control status. Descriptive statistics (DS) including percentage, mean ± standard deviation was calculated. Student's t-test and Mann–Whitney's U test were used for comparisons of continuous variables. Multiple logistic regression models were used to test for the interaction between various environmental risk factors.
MT in 525/1,075 cases all mutations preferentially occurred in exons 8 and 10 of FGFR2, FGFR3, FGFR4, TWIST, and MSX genes encoding the third Ig loop of the receptor in CS cases. FGFR2 mutations that we identified as CS + NSCS group including three missense substitutions causing CS 2 bp deletion. A premature stop codon and producing JW phenotypeFGFR2 mutations were selected in both the groups of CS + NSCS. Tyrosine kinase subdomains in the Ig I loop have FGFR2 mutations creating cysteine residues (W290C and Y340C) responsible for PS. Other residues amino acid (W290G/R, Y340H) located in Crouzon and the Apert phenotype. FGFR2 mutations in CS and PS were wider genotype–phenotype analyzed through gene testing. PS and CS had distinct sets of FGFR2 mutations. Hence, we also limited the number of recurrent amino acid changes verify among W290C, Y340C, C342R, and S351C is associated with Pfeiffer phenotypes.
As a novel intragenic mutations (IGMs) and deletions were involved in our Indian patient's samples using a new strategy to screen for FGFR family (1, 2, 3, 4), TWIST, MSX mutations to verify all odds and doubt cases. We used PCR amplification having subsequent CS + NSCS sequencing. We identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis identifies larger deletions adding an encompassing with the several genes. Microsatellite and fluorescence in situ hybridization (FISH) study reported that deletions are located in gene. The gene dosage assay (GDA) with “PCR walking” across with the critical region among 525 patients with Indian children with CS. All the features including with Apert, Saethre–Chotzen's syndrome (SCS) and other different non syndromic clinical conditions are observed carefully. Ten per cent were detected deletions by real-time gene dosage analysis patients within translocation or inversion site. Therefore, at least 260 kb 3′ of the gene suggested clearly that they had position-effect mutations between both groups verified carefully in our laboratory for CS only (cases and control) through CS OPD, AIIMS, NewDelhi-29.
A total of 525 out of 1,075) patients with classic features of Apert, Crouzon, metopic, coronal, and SCS have DR for FGFR family 1, 2, 3, 4 and TWIST mutations was 75%. Therefore, the associated risk for developmental delay (DD) and IQ in patients with deletions involving the FGFR1, 2, 3, 4 and TWIST genes was approximately 95% or nine times more common in patients with IGMs. CS characterized by craniofacial and limb anomalies and IGMs of the TWIST gene 7p21 have been reported. As a cause of this disorder phenotypic overlap within all of CS syndromes and IGMs in FGFR2 and FGFR3,4 been demonstrated in our laboratory. There were complete gene deletions and missense pattern of FGFR (1, 2, 3,4) and TWIST. As of patients with SCS investigated earlier, patients clinically identified significant proportion. Phenotype, patients with CS affected with developed in metropolitan to rural sector creating a global burden. SCS phenotype and achondroplasia always carry the FGFR3 P250R mutation. We found heterozygous for different novel mutations in the coding region of FGFR (1, 2, 3, 4) and TWIST genes. But surprisingly, we have a deletion of only lesser copy of the entire TWIST gene. But FGFR had more than 450 novel mutation detected in both cases. This is a clue for our newly investigation of developmental delay among CS and NSCS, distinguishing feature of the patients with deletions, compared with patients with IGMs of FGFR and TWIST genes. The 85% with the Apert, Crouzon, metopic, coronal, and Saethre–Chotzen phenotype had detectable genetic changes through in FGFR (1, 2, 3, 4 and TWIST) with CS. But in real and routine life, NSCS development is higher, a great challenge now for India as well as for the world.
CS and NSCS are heterogeneous in terms of its causes, presentation, and management. Bicoronal and coronal synostosis evidences give a higher tendency and genetically caused in between. TWIST1 and FGFR3 identified as a major causative genes analyzed coronal synostosis by the clinical and molecular characteristics of Korean and Indian.
Coronal synostosis population mutation hot spots have the negative samples were subsequently screened for FGFR2 and TWIST1 mutations were detected p.P250R. Mutation in FGFR3 in bicoronal cases were evidenced a much higher mutation DR (MDR is 52.9%) than unicoronal cases (7.7%) reported by a researcher. TWIST1 group had SCS, and no syndromic group FGFR3 group, patients had Muenke's syndrome among the NSCS. A majority of the associated anomalies were exception of psychomotor retardation and Chiari malformation.
TWIST1 and FGFR3 p.P250R patients with CS in Indian population we have already started. The best of our knowledge, for the first time, we illustrate the frequency and spectrum of mutations in TWIST1 and FGFR (1, 2, 3, 4) genes in Indian population having both CS + NSCS. Considering this appropriate molecular diagnosis techniques, we prove that it is helpful in providing adequate genetic counseling, guidance, genetic screening for FGFR1, 2, 3, 4 and TWIST1 in cases with coronal synostosis. FGFR family having a very higher number of involvement rather than researcher reported terribly regarding MTHFR and other gene also involved in the CS and NSCS children.
We strongly proposed that initial screening for the FGFR3 P250R mutation, followed by sequencing of FGFR1, 2, 3, 4 and TWIST and then FISH for deletion detection rate (DDR) of TWIST genes are in sufficient to detect mutations in >90% of patients with the Apert, Crouzon, metopic, coronal, and Saethre–Chotzen phenotype as well. Mutation in SCS alterations has both proliferation and osteoblast gene expression in human calvarial osteoblasts (HCO), indicating that TWIST is an important regulator of osteoblast differentiation. We showed that TWIST haploinsufficiency altered the osteoblast apoptosis (OA) in SCS cases earlier. But as per analysis of terminal deoxynucleotidyl transferase-mediated nick-end labeling demonstrated that increased osteoblast and osteocyte apoptosis in coronal sutures from SCS patients with nonsense mutations favoring with (Y103X and Q109X).
Presently, bHLH-truncated proteins, and one patient with a missense mutation (MM) in the basic domain (R118C) that abolishes Twist DNA binding and this assessment in the mechanisms involved OA in the mutant (M-Tw). Therefore, it is very clear that calvarial cells bearing the Y103X mutation resulting in decreased TWIST mRNA. However, protein levels and M-Tw cells cultured in low serum conditions enhances DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Clinically, biochemical analysis increased the activity of initiator caspases-2 and -8. Downstream effector caspases were -3, -6, and -7 in mutant with osteoblasts.
The caspase-2 upstream of caspase-8 and effector caspases-3, -6, and -7 activities were suppressed by a specific caspase-2 inhibitor. Moreover, M-Tw osteoblasts increased cytochrome-c released from the mitochondrial activity on the downstream effector. The caspase-9 was not increased due to over expression on the antagonist protein Hsp70. Differentially expressed genes using by cDNA expression array revealed that Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells were increased findings were confirmed through RT-PCR and western blot analyses. Neutralization of TNFalpha overexpression using anti-TNFalpha or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8, caspases-3, -6, and -7, and M-Tw osteoblasts. Novel evidence added that TWIST haploinsufficiency in Apert, Crouzon, metopic, coronal, and SCS promotes OA through TNFalpha-caspase-2, caspase-8, caspases-3, -6, and -7 cascades. Molecular mechanism in which TWIST plays an antiapoptotic role in HCO. FGFR family (FGFR1, 2, 3, 4) having FGFR2 having significant role for reproducing these activities aggressively for both the (CS + NSCS) groups which give a better clue for targeting molecular therapy and targeting molecular medicine for future application.
Characterization and identification
CS and limb anomalies were screened for mutations in FGFR1, FGFR2, FGFR3, and FGFR4 and TWIST mutations were found among the larger number of families. Therefore, many families were found FGFR3 P250R mutation. FGFR2 VV269-270 deletion DR for TWIST or FGFR mutations is 75% in our Apert, Crouzon, metopic, coronal, and SCS; patients reported with FGFR mutations and TWIST mutations are also responsible in NSCS. In the world literature, most common phenotypic features, present in more than one-third of our patients with FGFR and TWIST mutations, are coronal synostosis, brachycephaly, low frontal hairline, facial asymmetry, ptosis, hypertelorism, broad great toes with clinodactyly. Still, significant intra- and interfamilial phenotypic variabilities between both groups in TWIST mutations and FGFR mutations overlap in clinical features and the presence of phenotypic abnormalities. As of mutations rates are more than one, craniosynostotic condition—such as Saethre–Chotzen, Crouzon, metopic, and Pfeiffer syndromes—supports the hypothesis that TWIST and FGFRs strict to these conditions.
Gene expression, tissue engineering, and clinical practice
The components of the molecular pathway involved in the modulation of craniofacial and limb development in humans. Craniosynostosis syndromes are Apert, Crouzon, Pfeiffer, Jackson– Weiss, and Crouzon syndrome with acanthosis nigricans. FGFR genes unrelated patients with FGFR2-related with Craniosynostosis syndromes are screened for mutations study. In exons IIIa and IIIc of FGFR2 patient with craniosynostosis had significant association noted. Pfeiffer syndrome was found a novel G-to-C SSM at-1 relative to the start of exon IIIc. Mutations reported C1205G was unusual findings are observed in our study. However, in related patients, one with clinical features of Pfeiffer's syndrome and the other having mild Crouzon's syndrome has same degree of phenotypic variability shown 90%. Moreover, clinical features associated with a specific mutation do not necessarily breed true. CS, craniofacial dysmorphism, digital anomalies, umbilical, and a genital abnormalities mislead by AD craniosynostotic syndromes. Crouzon's, Jackson–Weiss', Pfeiffer's, and Apert's syndromes are reported in the FGFR2 extracellular domain. Even, Crouzon's syndrome patients with acanthosis nigricans mutation occurred in the transmembrane domain (TMD) of FGFR3 is still questionable in Indian point of view. Describing the detection of FGFR2 mutations in the Beare–Stevenson cutis gyrata syndrome in sporadic cases, a novel missense mutation (NMSM) found causing an amino acid to be replaced by a cysteine molecule. An identical Ty375Cys mutation in the TMD. Ser372Cys mutation in the carboxyl-terminal end of the LR between the Iglll and TMDs patients, neither of these mutations was found suggesting further genetic heterogeneity. FGFR2 extracellular domain. Crouzon's syndrome patients with acanthosis nigricans had recurrent mutation. It occurs in the TMD of FGFR (1, 2, 3, 4). The detection of FGFR2 mutations in the Beare–Stevenson cutis gyrata syndrome among sporadic cases, a NMSM. We found causing of an amino acid be replaced by a cysteine; many had the identical Ty375Cys mutation in the TMD and Ser372Cys mutation in the carboxyl-terminal end of the conserved linker region are more than 350/525 cases reported from our NSCS cases in our new findings. But in heterozygous mutation (HM) 95/525 non syndromic and syndromic cases are also find out in our study.
HM again repeated the old story for both SYCS and NSCS of Asian Indian children as a leading cause in our findings. Here, for the first time, we clearly reported that IgIII of FGFR2 mutations outside this region of the protein and tyrosine kinase (TK1 and TK2) responsible for both in molecular and cellular levels for CS. It adds an evidence for future molecular targeting therapy. The article summarized that although so many evidence-based approaches available for CS. Particularly, sample preparation, characterization, identification, gene expression, and tissue engineering are quite necessary. Pertaining to this study and further progress need special attention and we encourage collaborating with other researchers, who have a keen interest for further study for the benefit of patients taken as molecular mission and eradicate the disease as mission for this country and the worldwide ability to solve and strength the lives.
We thanks to our patient and their relatives for participating in our study.
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Conflicts of interest
There are no conflicts of interest.