Patient 1

This patient had partial colectomy at age 16 years. She has had iron deficiency anemia requiring blood transfusion likely due to poor nutritional intake as well as menorrhagia. She has thoracic and lumbar scoliosis and bilateral protrusio acetabuli. She has been noted to have thick calvarium prominent on both frontal regions on head magnetic resonance imaging. She gets recurrent episodes of nausea and vomiting and severe headaches. She complains of feelings of hypoglycemia which requires her to eat frequent meals throughout the day. She has had surgery on right foot due to upturning toes. She was noted to have ectopia lentis in the left eye requiring lensectomy and partial vitrectomy at age 17 years. She also has myopic macular degeneration. She had many features of Marfan syndrome which are listed in Table I. She was on oral contraceptives and was having regular menstrual periods. She was taking iron and vitamin D daily. Interestingly, her total body fat on dual emission X-ray absorptiometry was 28.4%. An echocardiogram at age 23 years demonstrated normal left ventricular ejection fraction, somewhat myxomatous appearing leaflets with no significant prolapse. Her aortic root diameter at the sinus of valsalva was 2.5 cm and at the sinotubular ridge was 2.0 cm (Z score: 1.56).

Patient 2

She reported mild strabismus of the left eye. She had weekly episodes of severe headaches. She also had some anxiety, depression, and attention deficit disorder and received medications for this. She had scoliosis and had pain in right hip. She also had mild hearing loss in left ear. She had menarche at age 16 years and was having regular menstrual periods. She had several features of Marfan syndrome which are listed in Table I. Her total body fat on dual emission X-ray absorptiometry was 27.7%. On ophthalmologic examination, she had extreme myopia and iridodonesis but there were no lens dislocation. A recent echocardiogram demonstrated a widened aortic root (diameter at the sinus of valsalva 3.1 cm and at the sinotubular ridge 2.4 cm; Z score: 2.29).

DNA was extracted from peripheral blood leucocytes from the patients and their relatives. Whole exome sequencing of the two patients was performed by Knome Inc. (Boston, MA) using the SureSelect Target Enrichment System for Illumina Pair-End Sequencing Library protocol. The mean depth of coverage of targeted regions for Patients 1 and 2 were 76.5 and 77.5, respectively. A total of 28,991 and 28,529 autosomal variants passing the GATK quality filters were called for Patients 1 and 2, respectively.

We filtered their variants by function, conservation and novelty, and then examined their intersection (Table II). After filtering by the predetermined criteria for potentially pathogenic variants, 105 remained in Patient 1 and 64 remained in Patient 2. There was not a single variant shared by both patients, but there were two genes, KLHL35 and FBN1, harboring novel heterozygous missense variants in both of them (Table III). Sanger sequencing of c.225C>A (p.Pro75Thr) variant in KLHL35 in Patient 1 and her parents revealed that the unaffected father also harbored the same variant and of c.1439G>T (p. Arg480Leu) variant in Patient 2 and her parents revealed her unaffected mother as a carrier of the variant, thus excluding KLHL35 variants as disease-causing.

Patient 1 had heterozygousc.8206_8027insA(p.Thr2736Asnfs*1) mutation and Patient 2 had two heterozygous variants in close proximity to each other, that is, c.8222T>C (p.Ile2741Thr) and c.8226+1G>T (c.IVS64+1G>T; predicted to result in p. Glu2742Glufs*43). These variants were confirmed upon Sanger sequencing (Fig. 1A–D). Furthermore, none of their parents and none of the two siblings of Patient 1 and the five siblings of Patient 2 harbored any of these variants suggesting that these mutations were de novo. Interestingly, both the heterozygous variants in Patient 2 were on the same allele (Fig. 1E). There was no other variant in FBN1 present in either patient.

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FIG. 1.

Chromatograms from our Patients with progeroid fibrillinopathy. A: Electropherogram for normal sequence of a part of exon 65 of FBN1 and B: the sequence from Patient 1 showing heterozygous c.8206_8027insA mutation. C: Normal sequence electropherogram of a part of exon 65 of FBN1 and D: the sequence from Patient 2 showing heterozygous c.8222T>C and heterozygous c.8226+1G>T mutations. The amino acid substitution is shown below the electropherogram. E: Data from whole exome sequencing of Patient 2 in the region of exon 65 of FBN1 showing that both the heterozygous variants c.8222T>C and c.8226+1G>T are on the same allele.

Our data support previous observations that de novo FBN1 null mutations in patients presenting with manifestations fitting Marfan syndrome but also progeroid features are disease-causing [Graul-Neumann et al., 2010; Goldblatt et al., 2011; Horn and Robinson, 2011; Takenouchi et al., 2013]. Our study excluded the possibility of disease-causing variants in other genes (both de novo variants and homozygous or compound heterozygous variants) and thus provides firm evidence linking FBN1 variants with this syndrome. Furthermore, the variants in FBN1 in our patients and those reported previously are in close proximity. The splice site mutation c.8226+1G>T was previously reported [Horn and Robinson, 2011] and the frameshift insertion c.8206_8027insA seemed to be deposited into dbSNP 136 (rs193922241). All the variants are null variants either due to a nucleotide insertion, deletion or splice site alteration. This suggests clustering of null variants near exon 65 of FBN1 causing a phenotype strongly resembling Marfan syndrome with additional progeroid features. We suggest calling it progeroid fibrillinopathy. The pathogenic significance of the de novo heterozygous missense variant, c.8222T>C (p.Ile2741Thr), which is on the same allele as the other heterozygous null c.8226+1G>T variant in Patient 2, is not clear. The c.8222T>C variant is not a known polymorphism; however, PolyPhen-2 in silico prediction of its function was benign. Previously, a few individuals with other syndromes harboring two nearby de novo missense mutations on the same allele have been reported [Tessitore et al., 1999; Rump et al., 2006; Margraf et al., 2012].

FBN1 contains 66 exons and encodes for fibrillin-1, a 350-KDa, 2871-amino acid glycoprotein [Jensen et al., 2012]. Although collagens, laminins, and fibronectin are the main protein components of the extracellular matrix, fibrillins act as major structural components as 10–12 nm calcium-binding extensible microfibrils. The microfibrils provide a scaffold for elastin in elastic issues. While Marfan syndrome patients have mutations in FBN1 scattered all throughout the gene [Detaint et al., 2010], clustering of mutations in and around exon 65 have been responsible for the progeroid fibrillinopathy until now (Fig.2). Takenouchi etal.[2013] proposed that the presence of an extremely charged “ETEKHKRN” protein motif in the carboxyltermini of the truncated FBN1 proteins may be responsible for this severe phenotype. However, mutated FBN1 variants in both our patients lack this “ETEKHKRN” motif and thus our data do not support this hypothesis.

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FIG. 2.

The gene structure of FBN1 at the 3′ end and clustering of the reported de novo variants in and around exon 65 in patients with progeroid fibrillinopathy. Boxes denote exons and the lines between the two boxes denote introns. Shaded area denotes untranslated region of the exon. The c.8155_8156delAA mutation was previously reported by Graul-Neumann et al. [2010], c. 8156_8175del mutation by Goldblatt et al. [2011], c.8226+1G>T mutation by Horn and Robinson [2011], and c.8175_8182del mutation by Takenouchi et al. [2013]. The total number of exons in the FBN1 transcript is 66 and the first exon is noncoding.

ACKNOWLEDGMENTS

REFERENCES