Abstract
Background Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder characterized by skeletal abnormalities such as hypoplasia of the mandible and clavicles and acro-osteolysis. Other features include cutaneous atrophy and lipodystrophy. Two genetic loci are known for MAD: lamin A/C (LMNA), encoding structural nuclear lamina proteins, and zinc metalloproteinase (ZMPSTE24), a membrane-bound endoprotease involved in post-translational proteolytic cleavage of carboxy terminal residues of prelamin A to form mature lamin A.
Methods Mutational analysis of ZMPSTE24 in an additional patient with MAD and determination of functional activity of mutant ZMPSTE24 in a yeast growth arrest pheromone diffusion (halo) assay.
Results We previously reported a Belgian woman with MAD who had ZMPSTE24 mutations and died of complications of chronic renal failure at the age of 27.5 years. We now report a 37-year-old Australian man with MAD who also had compound heterozygous mutations in the ZMPSTE24 gene, a null mutation, Phe361fsX379, and a missense mutation, Asn265Ser, which is partially active in the yeast complementation assay. He also developed end-stage renal disease and, despite receiving a cadaveric renal transplantation, died prematurely at the age of 37 years. Renal biopsies of both patients revealed focal segmental glomerulosclerosis, and the female patient had the collapsing variant.
Conclusion These observations suggest focal segmental glomerulosclerosis as a phenotypic manifestation in patients with ZMPSTE24 deficiency.
Mandibuloacral dysplasia (MAD; Online Mendelian Inheritance in Man #248370 and 608612) is a rare autosomal recessive syndrome with variable clinical features, including mandibular and clavicular hypoplasia, acro-osteolysis of terminal phalanges, delayed closure of cranial sutures, joint contractures, mottled pigmentation, cutaneous atrophy, and lipodystrophy. MAD is phenotypically and genetically heterogeneous, and two loci, LMNA, which encodes integral nuclear lamina proteins lamins A and C, and ZMPSTE24, which encodes a zinc metalloproteinase involved in post-translational processing of prelamin A, have been identified. So far, 21 MAD patients have been reported to have mutations in LMNA, 1-6but only 2 patients have been reported with compound heterozygous mutations in ZMPSTE24. 7,8One of them, a young Belgian woman, had some peculiar clinical features not noted in others with the LMNA mutations, such as early onset of MAD, generalized lipodystrophy, severe progeroid features, bony knots on phalanges, and progressive glomerulopathy, 7,9and she died of complications of chronic renal failure at the age of 27.5 years. 7The other patient had additional features of progeria and restrictive dermopathy and died prematurely at the age of 2 years, 9 months. 8We now report compound heterozygous mutations in ZMPSTE24 in another patient with MAD who also had end-stage renal disease requiring renal transplantation. 10,11We also report focal segmental glomerulosclerosis (FSGS) in both adults with ZMPSTE24 deficiency.
CLINICAL FEATURES OF THE PATIENTS
The studies were approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center at Dallas, Texas; University of Melbourne, Melbourne, Victoria, Australia; and University Hospital of Leuven, Leuven, Belgium, and written informed consent was obtained from patient 1 and from the legal guardians of patient 2.
Patient 1
The clinical features of this patient (MAD 600.5) have been reported. 7,9Briefly, she had a small chin, nose, and mouth and thin facial skin at birth. She had delayed closure of the cranial sutures and at the age of 2 years developed acro-osteolysis. Many bony knots were noted on the hands and toes, requiring surgical removal. The histology of these lesions revealed sclerotic tissue with calcification. She had menarche at age 15 years but had scarce secondary sexual characteristics. She developed joint contractures in the hands and had scarce and brittle hair. She had lipodystrophy affecting her face and extremities. The renal disease was discovered at the age of 25 years when she was hospitalized for sepsis and acute respiratory distress owing to pulmonary infection. She developed anuria and had serum urea concentration of 200 mg/dL and creatinine concentration of 6 mg/dL. Hemodialysis was initiated, and renal biopsy was performed after stabilization of her condition. She underwent chronic peritoneal dialysis until her death at age 27.5 years owing to pulmonary edema and malignant hypertension.
Patient 2
The clinical features of this patient were published first at the age of 6 years 10and then again at the age of 29 years. 11Briefly, he was a child of nonconsanguineous parents who presented with widely open cranial sutures at 18 months of age, protuberant eyes, a narrow nose, marked maxillary midline hypoplasia, and micrognathia with unique mandibular midline dysplasia involving both the bone and incisor teeth. He had relatively short limbs, a narrow chest with underdeveloped clavicles, delayed ossification of the pubis, short and broad fingers, and fixed flexion deformities of his knees with patchy areas of atrophic pigmented skin. Progressive severe kyphoscoliosis developed that required surgery. He also had calcified necrotic ulcers on his scalp, ears, elbows, and fingers, with resorption of the tips of his fingers. He had retarded growth and developed obstructive sleep apnea requiring tracheostomy at the age of 18 years. At the age of 19.5 years, he was noted to have hypertension, microscopic hematuria, and nephrotic-range proteinuria, which led to a renal biopsy. His serum creatinine level was 1.2 mg/dL. Renal pathology revealed FSGS with extensive tubular atrophy and interstitial fibrosis. 11He developed hypertensive encephalopathy with seizures followed gradually by renal failure at age 26 years, which was treated by hemodialysis. At the age of 29 years, he underwent successful cadaveric renal transplantation.
Over the next 5 years, he suffered from chronic ischemic leg ulcers secondary to severe calcific peripheral vascular disease and from recurrent thoracic subcutaneous abscesses, which were complicated twice by staphylococcal septicemia. Subtotal parathyroidectomy was finally performed at age 31 years. Despite renal transplantation, the hypertension remained difficult to control. He underwent coronary angioplasty for chest pain at age 31 years. He also developed multiple squamous cell carcinomas and basal cell carcinoma on his nose.
Five years after renal transplantation, he developed progressive weakness and spasticity in both the legs and neurogenic bladder owing to spinal cord compression at thoracic vertebrae 11 and 12. He developed osteomyelitis of thoracic vertebrae 11 and 12 at age 35 years. Additional problems included diabetes mellitus, deafness secondary to vancomycin, progressive soft tissue calcification, pressure ulcers, and progressive flexion deformity of the hands. By age 36 years, he was mostly confined to bed and died of septic shock at the age of 37 years secondary to chronic osteomyelitis.
METHODS
Mutational Analysis
Genomic deoxyribonucleic acid (DNA) from patient 2 and his parents was isolated from the blood sample using DNAzol (Gibco, Gaithesburg, MD) according to the manufacturer's protocol. The exons and splice-site junctions of the ZMPSTE24 gene were amplified as described earlier. 7The polymerase chain reaction (PCR) product was purified to remove primers and deoxy dinucleotide triphosphate (dNTPs) and sequenced using ABI Prism 3100 (PE Applied Biosystems, Foster City, CA). Sequences were compared using Vector NTi Suite 6 software (Informax, Bethesda, MD) and by visual inspection.
ZMPSTE24 Mutant Construct
To create the Asn265Ser mutant, the plasmid pRS416GPD-hZMPSTE24-wt containing the ZMPSTE24 complementary DNA was used as the template with the following primers: 5′-TCCCACAGCAGTGCTTATTTTTATG-3′ (sense) and 5′-CATAAAAATAAGCACTGCTGTGGGA-3′ (antisense). 7The mutation was generated using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA) as suggested by the manufacturer. The resulting yeast expression vector pRS416GPD-hZMPSTE24-Asn265Ser was again sequenced to ensure that there were no PCR errors.
Growth Arrest Pheromone Diffusion (Halo) Assay
To determine if the missense ZMPSTE24 mutation was active in complementing the yeast STE24, the yeast expression plasmid pRS416GPD-hZMPSTE24-Asn265Ser was transformed into yeast strain 3614 (MATαste24Δrce1Δ) and grown on selective medium. Liquid cultures were concentrated, and 1 μL aliquots were then spotted on to a lawn of mating yeast (strain XBH8-2C, MATα) on Yeast extract, peptone and dextrose (YPD) medium with 0.043% Triton X-100 12and allowed to grow for 2 days at 30°C. The production of mature a-factor is proportional to the zone of growth inhibition (halo) formed. Also included in the assay were the expression vectors for the wild type, pRS416GPD-hZMPSTE24-wt, and the null mutant, pRS416GPD-hZMPSTE24-Phe361fsX379, as reported earlier. 7
Histology and Immunohistochemistry
These studies were limited by the availability of the tissues and pathology slides. We received several slides, along with a small piece of frozen tissue from a renal biopsy from patient 1. We also received slides from patient 2. The slides were reviewed, and the frozen tissue was used for immunohistochemistry.
RESULTS
Patient 2 had compound heterozygous mutations in ZMPSTE24. One of the mutations causes a frameshift and a premature termination codon, resulting in a truncated protein, Phe361fsX379, and was reported in our Belgian patient. 7The second mutation was a missense mutation, at nucleotide 794 A>G, which results in substitution of asparagine residue at position 265 to serine. The asparagine at position 265 is conserved among the species examined (Figure 1). This mutation was also noted in the young girl described by Shackleton and colleagues. 8
The functional significance of the missense ZMPSTE24 mutation was assessed in the yeast complementation assay. In this assay, the secreted mature a-factor diffuses into the medium and arrests the growth of MATα strain, leaving a “halo” surrounding the MATα strain. The size of the halo is proportional to the production of mature a-factor. Figure 2 shows the results of the yeast halo assay assessing the complementation of the a-factor processing defect by transforming the ste24Δrce1Δ yeast with expression vectors for the wild-type ZMPSTE24 or the mutations found in our subject. The expression vector containing human ZMPSTE24 (wild type) restored mature a-factor production and resulted in growth arrest of MATα cells, as judged by the halo surrounding the colony. The ZMPSTE24 mutant, Asn265Ser, was only slightly active in complementing the yeast a-factor and produced a smaller halo around the colony (see Figure 2). As reported previously, 7the null mutant, Phe361fsX379, was totally inactive (see Figure 2).
The renal biopsy from patient 1 contained 24 glomeruli, 21 of which were globally sclerotic. Of the three remaining glomeruli, two showed global and one demonstrated partial collapse of the tufts, with marked overlying podocyte hypertrophy and hyperplasia. There was marked tubular atrophy and interstitial fibrosis with a mild to moderate degree of interstitial lymphocytic infiltrate. Some tubules were dilated with proteinaceous casts. The interlobular arteries disclosed significant intimal fibrosis and muscular hypertrophy. The arterioles revealed severe hyalinosis. Immunoperoxidase studies were performed on frozen tissue that contained up to five glomeruli, all of which were globally or nearly globally sclerotic. Staining with immunoglobulin (Ig)G, IgA, and C1q was negative. The sclerotic glomeruli showed segmental staining for IgM and C3. There was no amyloid deposition. Taken together, the morphologic features showed FSGS, collapsing variant (Figure 3).
The renal biopsy of patient 2 contained 35 glomeruli, 25 of which were globally sclerotic. Seven glomeruli displayed segmental sclerotic lesions. There was marked tubular atrophy and interstitial fibrosis with significant lymphoplasmocytic infiltrate. The interlobular arteries and arterioles demonstrated significant muscular hypertrophy. Immunohistochemistry studies revealed segmental mesangial IgM staining. Thus, the morphologic features showed FSGS.
DISCUSSION
There seems to be an association between ZMPSTE24 deficiency and renal disease in humans as both adult patients with MAD so far known to have compound ZMPSTE24 mutations developed end-stage renal disease, which contributed to their premature death. Furthermore, there were interesting similarities in the renal histopathology and both of them showed FSGS. However, Zmpste24 −/− mice did not reveal abnormal renal function, although the renal histomorphologic features were not reported. 13,14Pendas and colleagues observed a strong expression of Zmpste24 in the epithelial cells of proximal and distal tubules and the collecting ducts of the kidney. 13In Zmpste24 −/− mice, serum creatinine levels were normal at the age of 8 weeks and at the end stage (24 weeks) of life. 13The tissue extracts from the kidney did reveal the lack of a 72 kDa lamin A band and the presence of a 74 kDa prelamin A band on Western blotting. Bergo and colleagues also did not mention any renal phenotype in their Zmpste24 −/− mice. 14Interestingly, neither patient had any evidence of dilated cardiomyopathy or muscular dystrophy, features noted in Zmpste24 −/− mice. 13,14
The renal biopsy sample of patient 1 showed collapsing glomerulopathy, a subtype of FSGS, and patient 2 had classic FSGS. Podocytes are the cells mainly affected in FSGS, such that swelling, hyperplasia, and cytoplasmic basophilia are frequently observed. 15In the collapsing variant, marked proliferation and hypertrophy of visceral epithelial cells occur with glomerular tuft collapse. In collapsing glomerulopathies, striking abnormalities of the podocyte architecture, with loss of foot processes, primary processes, and the actin-based cytoskeleton, have been observed with the progression of FSGS. 16FSGS can also be caused by advanced renal disease, with a reduction in functioning nephrons. However, patient 2, whose renal biopsy showed classic FSGS, had only mild renal disease, with a serum creatinine level of 1.2 mg/dL at the time of renal biopsy, Furthermore, advanced renal disease may not explain the collapsing glomerulopathy that occurred in patient 1 as it is generally believed that collapsing glomerulopathy is not associated with a reduced renal mass.
FSGS has many etiologies, primary or secondary. At least four genetic loci, namely nephrin (NPHS1), podocin (NPHS2), CD2-associated protein (CD2AP), α-actinin-4 (ACTN4), and canonical transient receptor potential 6 (TRPC6), have been associated with FSGS as a purely renal disease. 17-19The other loci, such as the Wilms' tumor gene (WT1), mitochondrial transfer ribonucleic acid (Leu), and SW1/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1 (SMARCAL1), are associated with extrarenal manifestations besides FSGS. 20-22We now report a new genetic locus, ZMPSTE24, associated with the collapsing variant of FSGS. Whether ZMPSTE24 deficiency primarily affects podocytes or epithelial cells of the glomerular tuft, or both, remains to be determined.
Another interesting clinical feature in both patients was the presence of “bony knots” on the phalanges that contained sclerotic tissue with calcification. In fact, the report of bony knots along with MAD in patient 2 at an early age aroused the initial suspicion of ZMPSTE24 deficiency. Interestingly, bony knots have not been reported in any of the 13 patients with the homozygous missense mutation Arg527His in LMNA. 1-3Thus, the presence of premature renal disease and bony knots on the phalanges may help in clinically differentiating between the two genotypically heterogeneous varieties of MAD.
The mutation Asn265Ser found in patient 2 involves asparagine, a residue well conserved across many species. In the yeast halo assay, the ZMPSTE24 Asn265Ser mutant construct was only partially active, as was the previously studied missense mutation, Trp340Arg, observed in patient 1. The other mutation, Phe361fsX379, has been shown to be totally inactive. 7The overall results do indicate that both patients had ZMPSTE24 deficiency. Unfortunately, a lack of available tissue samples for Western blotting from these patients did not allow us to demonstrate to what extent prelamin A is processed in these two patients.
ZMPSTE24 deficiency seems to be associated with variable phenotypes, such as MAD, progeroid syndrome, restrictive dermopathy, and FSGS. 7,8,23,24Prelamin A remains as yet the only recognized substrate of ZMPSTE24 in mammals. The precursor of lamin A is first synthesized as a 74 kDa precursor, prelamin A. It has been recently confirmed that ZMPSTE24 is involved not only in the proteolysis of the three C-terminal residues of the CAAX-box after farnesylation and methylation of the cysteine residue but also in the second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. 25Therefore, the clinical manifestations of the affected patients with ZMPSTE24 mutations may be attributed to defective processing of prelamin A. Type A lamins interact with many proteins, including nesprin 1α, emerin, lamina-associated polypeptides 1C and 2α, and actin. 26Disruption of interaction of lamin A with actin may lead to podocyte cytoskeleton derangement and FSGS. Alteration of regulation of the actin cytoskeleton of glomerular podocytes is considered to be the cause of FSGS in patients with mutations in ACTN4. 27However, it is very likely that ZMPSTE24 may also be involved in proteolytic processing of other CAAX-motif proteins that have not yet been identified. Some of these proteins may be predominantly expressed in the podocytes or visceral epithelial cells of the glomerular tuft, and their defective processing may result in the collapsing variant of FSGS in patients with ZMPSTE24 mutations.
ACKNOWLEDGMENTS
We thank the members of the families for their invaluable contribution to this project, Drs. S. Michaelis for yeast strain SM3614, B. Horazdovsky for yeast expression vector pRS416GPD and mating strain XBH8-2, and R. Auchus for help with the yeast halo assay. We also thank Drs. M. Finlay, The Royal Melbourne Hospital, and B. Van Damme and E. Lerut, The University Hospital of Leuven, for assistance with access to archived tissue. We acknowledge R. G. Huet and J. Sprayberry for management of the DNA and patient databases, illustrations, and technical assistance.