Keywords
Marfan syndrome - novel variation - next-generation sequencing
Introduction
Marfan syndrome (MFS) (Online Mendelian Inheritance in Man or OMIM#154700) is an autosomal
dominant inherited connective tissue disorder affecting cardiovascular, ocular, skeletal
systems with a prevalence of one of 5,000 to one of 10,000 cases.[1] Life-threatening cardiovascular system findings include aortic root aneurysm, mitral
valve prolapse with/without regurgitation, tricuspid valve prolapse, enlargement of
the proximal pulmonary artery, and dilatation of the aorta. Ocular findings are myopia,
ectopia lentis, glaucoma, cataract, and increased risk of retinal detachment. Skeletal
system findings include disproportionately long extremities for the size of the trunk
(dolichostenomelia), joint laxity, pectus excavatum or pectus carinatum, and scoliosis.
Clinical diagnosis based on the “Ghent nosology'' was revised in 2010.[2]
FBN1 (OMIM *134797) gene, located on 15q21.1, is responsible for Marfan syndrome. FBN1 is a relatively big gene containing 65 exons and it encodes a 350 kDa glycoprotein
called fibrillin.[3] Fibrillin protein controls the stability of extracellular microfibrils. If there
is not a known family history of Marfan syndrome, the diagnosis should be made with:
An FBN1 pathogenic variant known to be associated with MFS AND one of the following:
The large clinical variability and other connective tissue disorders with similar
findings are confusing factors for MFS diagnosis. In the present study, three cases
are presented with Marfan syndrome.
Case Report
Case 1
A 13-year-old male was referred to our Medical Genetics Department from the Department
of Pediatrics with a prediagnosis of Marfan syndrome. He was presented with chest
pain, chest deformity, and myopia. The chest pain was present for 5 years and used
to last for approximately 5 minutes. There were no associated palpitations, autonomic
symptoms, presyncope or syncope, and the pain was not related to respiration or food
intake. When his family history was considered, his mother revealed that her mother's
two brothers were tall and thin.
On general examination, the patient was comfortable at rest, tall (1.75 m equivalent
to 97 percentile), and thin with arachnodactyly, pectus excavatum, dolichostenomelia,
positive wrist, and thumb signs, increased arm span/height, dolichocephaly, scoliosis,
downslanting palpebral fissures, malar hypoplasia, and keloid ([Figs. 1] and [2]). His total systemic score was 8 based on the revised Ghent nosology. [Fig. 1] demonstrates the patient's typical clinical signs of MFS. His arachnodactyly is
shown in [Fig. 3].
Fig. 1 Case one with typical Marfanoid habitus.
Fig. 2 Positive thumb sign of the case of one's father.
Fig. 3 Foot of case one with arachnodactyly.
Laboratory tests were within normal limits (full blood count, urea and electrolytes,
calcium, magnesium and phosphate, and liver function tests). Echocardiography showed
mitral valve prolapse, and aortic root Z-score was 0.23. Urinary ultrasonography showed hydronephrosis. Dynamic renal scintigraphy
detected nonobstructive left kidney with a mild stasis.
Genetic evaluation of the proband revealed a heterozygous NM_000138.4(FBN1):c.229G > A(p.Gly77Arg) likely pathogenic variation (PM1, PM2, PP2, PP3) on FBN-1
sequencing. After family evaluation, father and grandmother (father's mother) showed
Marfan syndrome characteristics. We detected the same variation in father and father's
mother.
Case 2
A 33-year-old female patient was referred with ascending aortic aneurysm and a prediagnosis
of Marfan syndrome. She had an aortic valve operation 2 years ago due to severe chest
pain and nausea. There was no positive family history except her mother and father
had hypertension.
Physical examination revealed a tall woman of thin habitus with arachnodactyly. She
had positive wrist or thumb sign, malar hypoplasia, striae on skin, and myopia. Her
total systemic score was 3 based on the revised Ghent nosology. Z-score was 12.26.
Laboratory tests were within normal limits (full blood count, urea and electrolytes,
calcium, magnesium and phosphate, and liver function tests). Echocardiography showed
functional artificial aortic valve, mild aortic regurgitation, and ascending aorta
graft.
Molecular analysis for Marfan syndrome was planned for this case; the result showed
pathogenic heterozygous novel NM_000138.4(FBN1):c.165–2A > G variation (in-silico analysis PVS1, PM2, PP3, and PS2). As the analysis
of parents showed no pathogenic variation, the case underwent de novo Marfan syndrome
diagnosis.
Case 3
Three year and 9 month-old-female patient consulted our clinical genetics department
with arachnodactyly and micrognathia. Her parents were nonconsanguineous and there
were no positive findings in the family history except nephrotic syndrome in her mother.
She was the first child of the family and mother had preeclampsia in the prenatal
period. She was born through cesarian-section in 38 weeks with a suspicious history
of meconium aspiration/asphyxia. Her birthweight was 2,850 g (10–25 p) and growth
parameters were mildly retarded. According to the parents she had aggression and used
to forget the names of the colors.
On examining the 3-year 9-month old patient, weight came out to be 15.7 kg (75–90
p). She had a broad forehead, bilateral epicanthus, prominent nose, retromicrognathia,
thin lips, pectus excavatum, and positive thumb sign. In echocardiography, she had
mitral valve prolapse and mild mitral insufficiency. Z-score was 0.5. Molecular analysis revealed a heterozygous novel NM_000138.4(FBN1):c.399delC(p.Cys134ValfsTer8) variation (in-silico analysis PVS1, PM1, PM2, PP3,
and PS2). This variation is associated with Marfan syndrome. We analyzed the parents,
but they both did not reveal the same variation. Our case was diagnosed as de novo
Marfan syndrome.
Molecular Analysis
After obtaining the informed consent form from the cases/families 2-mL EDTA (ethylenediaminetetraacetic
acid) peripheral blood samples were obtained from the patients. Isolation of deoxyribonucleic
acid (DNA) from peripheral blood samples was done using EZ1 DNA blood 200-µL isolation
kits (Qiagen, Hilden, Germany) in EZ1 Advanced XL (Qiagen, Hilden, Germany) nucleic
acid isolation device. DNA assay was performed with the Qubit dsDNA HS Assay Kit (Invitrogen).
Sequence analysis of 15 genes was performed using Qiaseq-Targeted DNA Panel Kit (CDHS-14630Z-997)
(Illumina). COL3A1, COL5A1, COL5A2, EFEMP2, FBN1, FBN2, NOTCH1, SKI, SLC2A10, SMAD2, SMAD3, TGFB2,
TGFB3, TGFBR1, TGFBR2 genes were analyzed. The variant analysis was performed by using Qiagen Clinical
Insight software.
Discussion
MFS is a connective tissue disorder including aortic root dilatation, ocular lens
dislocation, overgrowth of the long bones and chest deformity. Connective tissue disorders
have a wide variability of phenotypes. MFS-related disorders have similar symptoms
and thus, differential diagnosis should be done carefully. Our cases in this study
were diagnosed with suspected Marfan syndrome considering revised Ghent nosology and
molecular test results; the cases received the accurate diagnostic prognosis of MFS.
It has been reported that FBN1 gene mutations are the causes of MFS. Less than 10% of the patients with typical
clinical characteristics of MFS have TGFBR gene mutations.[5]
FBN1 is mapped to chromosome 15q21.1 and encodes a 2,871 amino acid protein. Pathogenic
variations of FBN1 may cause formation anomalies of fibrillin and microfibrils. FBN1 is expressed in different tissues and especially cardiovascular system, cornea, and
cartilage are affected with the mutations of the FBN1 gene.[3] FBN1 protein maintains microfibers and includes transforming growth factor-1 (TGF-1)
binding protein-like domains and calcium-binding epidermal growth factor-like domains
(cbEGF).
Missense FBN1 mutations are generally localized in cbEGF which disrupts the stability
of elastic fibers.[6] Patient one and patient two had missense mutations with MFS clinical features. Missense
mutations affect the structure of fibrillin-1 and disrupt the function.[7] Thus, missense mutations are leading to disorganized microfibrils and effect the
connective tissue.
TGFBR1, TGFBR2, and SMAD3 genes also affect the pathway of TGF-β, such as the FBN1 gene.[8] If there is dysregulation of TGF-β signaling, the risk of thoracic aortic diseases
increases. Cardiovascular diseases are the most significant clinical manifestations
of MFS and MFS-related disorders. Cardiovascular pathologies, such as aortic rupture
and aortic dissections may be life-threatening. The success of surgical management
in aortic diseases states the survival of patients.[9] In addition, education of patients about the symptoms and risks is significantly
important for taking care of themselves. Genetic counseling is important for these
patients or other cases who have a family history. Patient two had an aortic valve
replacement operation when she was 31 years old.
Mitral valve prolapse is another severe pathology in MFS. It is reported as mitral
valve prolapse is found in 40 to 54% of the patients with MFS.[10] Twenty-five percent of these patients have moderate to severe mitral regurgitation.
The variation of patient one was a missense mutation with rs794728290 the database
of single nucleotide polymorphisms (dbSNP) number. The predictions of this variation
were, mutation taster: disease-causing, Sorting Intolerant From Tolerant (SIFT): tolerated,
GERP (genomic evolutionary rate profiling) score: 5.1199, and DANN (deleterious annotation
of genetic variants) score: 0.9992. The same variation was found in his father and
father's mother with clinical findings of MFS. Autosomal dominant inheritance of MFS
was seen in this family; the family screening is crucial in MFS. There may be different
phenotypes within intrafamilial members with MFS as seen in this family. The father
of this case (patient 1) had ascending aorta enlargement and left ventricular type
1 diastolic dysfunction. He had an operation for chest deformity. Tissue healing was
difficult, so a graft was taken from his leg. The grandmother (mother of his father)
had chest deformity and cardiac valve pathology.
It is known that it is difficult to explain this complex genotype–phenotype correlation
of MFS. A novel and de novo missense variation was detected in patient two. Approximately
25% of the MFS patients have a de novo FBN1 pathogenic variant.[4] This variation is a A > G transition in splice site. The novel deletion in the FBN1 gene observed in our patient three was associated with MFS. Single base deletion
caused a stop codon in this pathogenic variation. Splice variant would possibly lead
to alternative splicing of Exon 3.
Although the phenotype–genotype relationship in MFS is not fully understood, pathogenic
variations are thought to predominantly affect the fusion of microfibrils.[11] There are experimental studies showing that fibrillin 1 is effective in achieving
tissue homeostasis rather than elastin formation.[11] Among the mechanisms causing the disease, decreased fibrillin-1 synthesis and pathogenic
variations of exons in the central region of the gene can be counted. This difference
also explains the clinical spectrum, which can range from severe neonatal Marfan to
isolated ectopia cordis.[12] The relationship between the various pathogenic variations and the phenotype is
insufficient and the clinical setting determines other risk factors specific to the
patient. The only exception to the weak correlation between genotype and phenotype
is neonatal MFS with a fatal course.
As FBN1 is a great gene, analyzing it with next-generation sequencing gives accurate and
cost-effective results in a short time. In this study, we analyzed 15 genes (COL3A1, COL5A1, COL5A2, EFEMP2, FBN1, FBN2, NOTCH1, SKI, SLC2A10, SMAD2, SMAD3, TGFB2,
TGFB3, TGFBR1, TGFBR2) to detect Marfan syndrome and other associated disorders. A patient associated with
arachnodactyly may be suspected with Marfan syndrome; after molecular analysis only
FBN2 variation may be detected in this patient and the diagnosis will be changed.
Both for cost-effectiveness and differential diagnosis, studying gene panels is preferable.
Conclusion
Our study added novel mutations of FBN1 gene to MFS clinical characteristics to the genotype–phenotype spectrum for the literature.
Molecular analysis has an important role in the accurate diagnosis of MFS and MFS-related
disorders. Correlation of clinical findings and molecular analysis will be helpful
for genetic counselling, prenatal diagnosis, and management of patients with the same
variations on the prognosis prediction.
