Lenz-Majewski Hyperostotic Dwarﬁsm with Hyperphosphoserinuria from a Novel Mutation in PTDSS1 Encoding Phosphatidylserine Synthase 1 Michael P Whyte,1,2 Amanda Blythe,2 William H McAlister,3 Angela R Nenninger,1 Vinieth N Bijanki,1 and Steven Mumm1,2 1
Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, St. Louis, MO Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, MO 3 Department of Pediatric Radiology, Mallinckrodt Institute of Radiology at St. Louis Children’s Hospital, Washington University School of Medicine, St. Louis, MO 2
ABSTRACT Lenz-Majewski hyperostotic dwarﬁsm (LMHD) is an ultra-rare Mendelian craniotubular dysostosis that causes skeletal dysmorphism and widely distributed osteosclerosis. Biochemical and histopathological characterization of the bone disease is incomplete and nonexistent, respectively. In 2014, a publication concerning ﬁve unrelated patients with LMHD disclosed that all carried one of three heterozygous missense mutations in PTDSS1 encoding phosphatidylserine synthase 1 (PSS1). PSS1 promotes the biosynthesis of phosphatidylserine (PTDS), which is a functional constituent of lipid bilayers. In vitro, these PTDSS1 mutations were gain-of-function and increased PTDS production. Notably, PTDS binds calcium within matrix vesicles to engender hydroxyapatite crystal formation, and may enhance mesenchymal stem cell differentiation leading to osteogenesis. We report an infant girl with LMHD and a novel heterozygous missense mutation (c.829T>C, p.Trp277Arg) within PTDSS1. Bone turnover markers suggested that her osteosclerosis resulted from accelerated formation with an unremarkable rate of resorption. Urinary amino acid quantitation revealed a greater than sixfold elevation of phosphoserine. Our ﬁndings afﬁrm that PTDSS1 defects cause LMHD and support enhanced biosynthesis of PTDS in the pathogenesis of LMHD. © 2014 American Society for Bone and Mineral Research. KEY WORDS: BONE TURNOVER; MATRIX VESICLE; OSTEOPETROSIS; OSTEOSCLEROSIS; PHOSPHATIDYLSERINE; PHOSPHOSERINE
Introduction enz-Majewski hyperostotic dwarﬁsm (LMHD)(1) is the ultrarare Mendelian craniotubular dysostosis (OMIM #151050)(2) reported by Sousa and colleagues(3) in 2014 to result from heterozygous mutation within the gene (PTDSS1) that encodes phosphatidylserine synthase 1 (PSS1). Whole exome sequencing was used to identify two de novo missense mutations in PTDSS1 carried among four unrelated LMHD patients.(3) A ﬁfth LMHD patient showed via Sanger sequencing a third heterozygous missense PTDSS1 defect.(3) Concomitantly, in vitro studies disclosed increased production of phosphatidylserine (PTDS) and that the LMHD mutations were gain-of-function.(3) Thus, LMHD represented the ﬁrst inborn-error of PTDS metabolism, although its further pathogenesis was unclear. Notably, PTDS binds calcium within matrix vesicles for nascent hydroxyapatite crystal formation(4) and enhances osteogenic differentiation of mesenchymal stem cells to promote bone formation.(5)
After Lenz and Majewski brieﬂy characterized LMHD in 1974,(6) reports of 7 additional patients helped establish the typical clinical manifestations,(7–14) and 3 further case reports described relatively mild forms of this disease.(15–17) All 11 patients represented sporadic LMHD.(6–17) LMHD features widely distributed osteosclerosis with expansion of the diaphyses of the long tubular bones together with erratic ossiﬁcation of the short tubular bones.(1,2) There is also growth restriction, a “progeroid” appearance, loose skin, and mental retardation.(1,2) Bone turnover marker (BTM) and histopathological studies of the LMHD skeleton have not been reported. We describe an infant girl with LMHD caused by a novel (fourth) heterozygous missense mutation (c.829T>C, p. Trp277Arg) in PTDSS1. BTMs suggested her hyperostosis and osteosclerosis resulted from accelerated bone formation. Urinary amino acid quantitation revealed hyperphosphoserinuria, explained perhaps by the increased PTDS biosynthesis caused by her PTDSS1 mutation.
Received in original form October 3, 2014; revised form October 28, 2014; accepted October 30, 2014. Accepted manuscript online November 1, 2014. Address correspondence to: Michael P. Whyte, MD, Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, 2001 South Lindbergh Boulevard, St. Louis, MO 63131, USA. E-mail: [email protected]
Additional supporting information may be found in the online version of this article. Journal of Bone and Mineral Research, Vol. 30, No. 4, April 2015, pp 606–614 DOI: 10.1002/jbmr.2398 © 2014 American Society for Bone and Mineral Research
Patient and Methods Patient The patient was referred to us in 1997 at age 51/2 months to investigate her skeletal disease. We studied her on that occasion only. She died at age 11 years. Prenatal ultrasonography, performed because her sister had Trisomy 21, had shown ﬁstlike hands. After 36 weeks gestation, birth weight was 2570 g (5th centile). Apgar scores were 7 and 8. Respiratory distress led to a 19-day stay in an intensive care unit and 14 days of home oxygen therapy. Enlargement of her anterior fontanel, nasal stufﬁness, and skin laxity were noted. Choanal stenosis was suspected but absent on computed tomography (CT). Renal ultrasound was normal at birth. Eye and hearing exams were unremarkable. Karyotype showed 46 XX. Antihistamine given to relieve nasal congestion allowed better breast feeding. LMHD was diagnosed during the ﬁrst month of life from her dysmorphic face and hands and loose skin and her radiographic changes evaluated by Dr Robert J Gorlin and Dr Leonard O Langer (University of Minnesota, Minneapolis, MN, USA). Her healthy mother and father were aged 35 and 36 years, respectively. At age 21/2 months, she required 2 to 3 days to fully recover from anesthesia administered for bilateral inguinal hernia repair. An atrial septal defect was diagnosed. By age 3 months, weight and height were clearly low. Her fontanel had become smaller. She had marked joint hypermobility and hyperlaxity of the skin that was fragile with prominent superﬁcial veins. An orthopaedist braced her hyperextended wrists. At age 51/2 months and after parental consent sanctioned by the Human Studies Committee at Washington University School of Medicine (St. Louis, MO, USA), the patient was admitted to the Center for Metabolic Bone Disease and Molecular Research at Shriners Hospital for Children in St. Louis, MO, USA (Research Center), where she continued her high-density formula (Similac Special Care 24 with Iron; Abbott Nutrition, Columbus, OH, USA) that provided 900 mg/d of calcium (RDA for age ¼ 210 mg).(18) Her development was slightly delayed with muscle weakness and joint hypermobility. Social development was appropriate. Her heart rate was 140 beats per minute without a murmur or gallop. She was small but active (length 58.6 cm [Z-score –2.3], weight 4.6 kg [Z-score –4.6], and head circumference 41.6 cm) (Fig. 1A). The anterior fontanel was enlarged, 9.5 12 cm, and sclera were blue. Her face showed several dysmorphic features (Fig. 1B). Nasal stufﬁness occurred without stridor, discharge, or signs of upper respiratory obstruction. The palate was not higharched. Two small lower incisors were present. Her very loose skin had no birthmarks (Fig. 1C). Abdominal surgical scars were not hypertrophic. There was no organomegaly. No gross deformity of the spine or major long bones was observed. However, her ﬁngers and toes were short, rendering the hands and feet small (especially distally) and deformed with hyperextension of all digits (Fig. 1D). Fingernails appeared large (Supplemental Fig. S1). Non-fasting blood and timed urine collections were studied. Leukocyte DNA was extracted using the Puregene DNA Puriﬁcation System (Gentra, Minneapolis, MN, USA) and then archived. Karyotype (46 XX) at St. Louis Children’s Hospital showed no abnormality of Giemsa-banded chromosomes (500 band stage). Her father was also investigated (mother unavailable). At age 6 months, degenerative retinal changes did not seem to impair her vision. A feeding tube was placed at age 7 months. She wore hearing aids. Choanal atresia was excluded by CT at
Journal of Bone and Mineral Research
age 8 months. Two months later, optic nerve “cupping” was concerning for foramen encroachment. At age 12 months, strabismus and mild optic pallor were documented. At age 13 months, her platelets were considered small, ineffective, and steadily declining. At age 15 months, difﬁculty eating occurred because of nasal obstruction and she lost weight. Nasal dilation was unsuccessful because of bony growth. A second attempt to remove bone led to severe postoperative bleeding. A sleep study showed desaturations requiring oxygen. Immunoglobulin was administered intravenously every 21 days, and she received hospice care. No further clinical information was available after 15 months. She died with no history of fracture in 2008 at age 11 years.
Radiological studies In 2014, we re-reviewed all of the radiological images provided to us in 1997.
Biochemical studies of mineral and skeletal homeostasis At the Research Center in 1997, we had evaluated the patient’s mineral homeostasis and, using commercial laboratories, her skeletal homeostasis by quantitating BTMs in non-fasting serum and timed urine collections. This included assay of serum bonespeciﬁc alkaline phosphatase (BAP; Metra Elisa Assay, Quidel Corporation, Mountain View, CA, USA) and osteocalcin (OCN; RIA method), as well as urinary amino acids including hydroxyproline (OHP) (Corning Nichols Institute, San Juan Capistrano, CA, USA) in one timed collection. In 2014, we assayed aliquots of three timed urine collections, frozen at –40°C since 1997, for free deoxypyridinoline (DPD Immulite 1000 Pyrilinks-D, Siemens Healthcare Diagnostics Products Ltd., Lianberis, Gwynedd, UK) “corrected” for urinary creatinine, and similarly tartrate-resistant acid phosphatase (Metra TRAP5b EIA kit; Quidel Corporation, San Diego, CA, USA) in serum. To explore for biochemical evidence of osteopetrosis,(19,20) serum creatine kinase (CK) and its isoenzymes had been quantitated in 1997 (Corning Nichols Institute).
Mutation analysis In 2014, each of the 13 coding exons and adjacent mRNA splice sites of PTDSS1 were PCR ampliﬁed and sequenced using the archived genomic DNA from the patient and father. Forward and reverse sequencing was performed for each exon. The primer pairs, designed in our laboratory, and annealing temperatures are provided (Supplemental Table S1). The sequencing electropherograms were examined visually and also aligned using Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA). The missense mutation was validated by its absence from the single nucleotide polymorphism database (dbSNP: www. ncbi.nlm.nih.gov/SNP).
Results Radiological findings Re-reviewed radiographs included the skeletal survey obtained during the ﬁrst 5 days of life and the chest ﬁlm from age 31/2 months (Supplemental Fig. S2). In the axial skeleton, the skull’s anterior fontanel was large. Osteosclerosis involved the base, petrous bones, and orbital roofs as well as the maxilla and mandible (Fig. 2A). The maxilla was slightly hypoplastic. Tooth development was delayed. The
Fig. 1. (A) Patient age 51/2 months. There is a disproportionately large head, mid-face hypoplasia, high-arching eyebrows, and apparently a small chest. (B) There is a cupid’s bow–like mouth with a downturned upper lip, seemingly large ears, small chin, prominent venous pattern of the scalp, and possibly some telecanthus. (C) There are hyper-rugated and redundant abdominal skin folds and prominent diastasis recti. (D) The severely malformed hand includes clinodactyly of digits 2 and 5 over 3 and 4 with short and disproportionately large ﬁngernails.
neonatal chest showed sclerotic clavicles that lacked medullary cavities and were widened at their medial two-thirds (Fig. 2B). The scapulae and ribs were uniformly sclerotic. The heart and lungs appeared normal. At age 31/2 months, the clavicles remained sclerotic and the medial two-thirds remained wide. Some periosteal new bone formation affected the left clavicle. The ribs were slightly less sclerotic and had become widened. All vertebral bodies and neural arches were uniformly sclerotic with no “rugger-jersey” appearance (Fig. 2C). The interpediculate distances were normal. The pelvic bones, too, were uniformly sclerotic with no “bone-in-bone” appearance (Fig. 2D). At birth, tubular long bones were uniformly sclerotic, including the metaphyses and epiphyses. At age 5 days, there
WHYTE ET AL.
was some metaphyseal and epiphyseal demineralization (Fig. 2E). There was minimal femoral and distal tibial bowing and some mild metaphyseal modeling defects with expansion especially in each radius. The olecranons were hypoplastic (Fig. 2F). The hands and feet were grossly abnormal (Fig. 2G). The ﬁngers were small. Metacarpals #1–4 were short, especially the 4th. No ossiﬁcation involved the 5th metacarpals. The phalanges of the thumbs were relatively well formed and ossiﬁed, except for minimal shortening of the proximal phalanx. The ﬁngers showed ulnar deviation and some syndactyly. The proximal and middle phalanges of the 2nd digits were fused and curved. The distal phalanx was reasonably developed. The proximal and middle phalanges of the 3rd digits were small. The 4th digits had
Journal of Bone and Mineral Research
Fig. 2. (A) The lateral radiograph of the skull shows the anterior fontanel is large and there is generalized osteosclerosis (particularly at the base, orbital roofs, and petrous bones, as well as at the maxilla and mandible). The maxilla is hypoplastic. There is delayed appearance of the teeth. (B) The clavicles are sclerotic and widened, particularly in the medial two-thirds. (C) The lateral newborn spine is diffusely osteosclerotic, including the vertebral bodies and neural arches. There is no “bone-in-bone” appearance. The ribs are slightly sclerotic but not widened. (D) The pelvis is sclerotic but not deformed. There is no “bone-in-bone” appearance. (E) The femurs, tibias, and ﬁbulas all are uniformly sclerotic, including the epiphyses, on the ﬁrst day of life. At age 5 days, some epiphyseal and metaphyseal demineralization appeared. Slight femoral and tibia bowing and slight metaphyseal expansion indicates a modeling error. (F) At birth, all tubular bones are osteosclerotic, with a modeling defect, especially of the distal radius. Both olecranon are hypoplastic. (G) In the left hand, metacarpals 1 through 4 are short, and the 5th is not ossiﬁed. There is fusion (symphalangism) and deformity of the second middle and proximal phalanges. There is asymmetric shortening and deformity of the other digits with symphalangism of the proximal and middle phalanges of the ﬁfth digit. The digits are not well ossiﬁed. (H) The feet are small with hypoplasia of metatarsals 1 to 4 bilaterally and no ossiﬁcation in the 5th metatarsals. The ﬁrst proximal phalanges are small and round. No other proximal phalanges are ossiﬁed. The distal phalanges are small, and there is soft tissue syndactyly. The talus and calcaneus are ossiﬁed bilaterally.
Journal of Bone and Mineral Research
a tiny proximal phalanx, a wide but short middle phalanx with tapering ends, and a more normal-size distal phalanx. The 5th digits bilaterally had fusion of a proximal and extremely hypoplastic middle phalanx, and a hypoplastic distal phalanx. Some overall sclerosis involved these bones. Symphalangism occurred between the proximal and middle phalanges of the 2nd and 5th digits and likely the 4th digits. In the feet, there was ossiﬁcation of the talus and calcaneus (Fig. 2H). The metatarsals were all short and slightly sclerotic. Progressing laterally, the metatarsals became more hypoplastic and the 5th metatarsals were not ossiﬁed. Only the 1st proximal phalanges were ossiﬁed, and these were round rather than tubular. Overall, the patient’s radiographic ﬁndings typiﬁed LMHD,(1) especially the changes in the hands and feet (Supplemental Fig. S2). CT of the patient’s head on the third day of life demonstrated marked areas of subcortical hypoattenuation in the frontal and parietal lobes. The base of the skull and facial bones were sclerotic (Fig. 3). The father’s lateral spine, hand, and skull radiographs were normal.
Biochemical studies of mineral and skeletal homeostasis Normal levels were found for serum sodium, 136 mmol/L (normal, 136–145); potassium, 5.0 mmol/L (normal, 3.6–5.3); chloride, 100 mmol/L (normal, 96–106); carbon dioxide, 24 mmol/L (normal, 22–31); albumin, 4.4 g/dL (normal, 3.4–5.2); total protein, 6.3 g/dL (normal 6.0–8.5); uric acid, 4.2 mg/dL (normal, 2.0–6.5); cholesterol, 122 mg/dL (normal, 50– 190); aspartate aminotransferase, 52 U/L (normal, 10–60); lactate dehydrogenase, 812 U/L (normal, 425–975); bilirubin, 0.5 mg/dL (normal, 0.2–1.2); blood urea nitrogen, 12 mg/dL (normal, 7–18); and creatinine, 0.4 mg/dL. While she continued her high-density formula, the patient’s mineral homeostasis seemed to reﬂect her high calcium intake with elevated serum calcium, 11.0 mg/dL (normal, 9.4–10.6) repeated and veriﬁed the subsequent day at 11.0 mg/dL; ionized
calcium, 5.4 mg/dL (normal, 4.9–5.4); magnesium, 2.4 mg/dL (normal, 1.8–2.3); and phosphorus, 6.6 mg/dL (normal, 4.0–5.9), although PTH (intact PTH: RIA-immunoradiometric assay, Nichols Diagnostics, San Juan Capistrano, CA, USA) was not suppressed but slightly elevated at 47 pg/mL (normal, 12–41), and three timed urine collections showed normal calcium/ creatinine ratios of 169, 106, and 97 mg/g and phosphorus/ creatinine ratios of 2.3, 1.6, and 1.3 g/g (Supplemental Table S2). BTMs revealed slightly elevated serum alkaline phosphatase (ALP) of 322 U/L (normal, 110–320), BAP of 187 U (normal, 34– 180 in our laboratory), and OCN of 49.1 ng/mL (normal for ages 2–17 years, 2.8–41) (Supplemental Table S2). Despite the disturbances in the patient’s skin and bones, urinary OHP was normal for age at 18.8 mmol/mole creatinine (normal, 0–23.7), urinary free DPD was 30.5, 40.5, and 44.4 nmol/mmol creatinine (normal, 10.5–45) with essentially identical urine creatinine concentrations compared with the assays performed in 1997, and serum TRAP5b was 14.0 U/L (normal, 6.3–26.7). In 1997, we had explored for possible osteoclast (OC) failure causing an osteopetrosis by quantitating serum total CK, which was normal at 107 IU/L (normal, 26–170), yet the CK isoenzyme quantitations suggested somewhat abnormal percentages with CK brain isoenzyme (CK-BB) 4% (normal, 0%), CK-MM 90% (normal, 97– 100%), and CK-MB 6% (normal, 0–3%) (see Discussion). The father had normal serum levels of calcium 9.3 mg/dL (normal, 9.1–10.3), ionized calcium 4.8 mg/dL (normal 4.8–5.4), phosphorus 3.5 mg/dL (normal, 2.4–4.4), ALP 63 U/L (normal, 29–115), BAP 30 U/L (normal, 2–38), and total CK 122 (normal, 26–170) with 0% CK-BB.
Urinary amino acid quantitation Urinary amino acid quantitation showed several elevated levels (Supplemental Table S3), but when the proﬁle was reviewed in 1997 by Dr Michael Landt (Inborn Errors of Metabolism Laboratory, St. Louis Children’s Hospital), the pattern was judged not consistent with a known disorder, with “nearly all of
Fig. 3. CT of patient’s head. (A) At age 3 days, the brain shows areas of marked subcortical hypoattentuation greatest in the frontal and parietal lobes. (B) The skull base and facial bones are sclerotic.
WHYTE ET AL.
Journal of Bone and Mineral Research
the high values within the normal range” for his laboratory. However, the phosphoserine level of 30.3 mM/M creatinine (normal for age, 0–4.0) seemed conspicuously elevated also with an excess of ethanolamine and slight increase of serine and phosphoethanolamine.
Mutation analysis Sanger sequencing revealed for our patient a single heterozygous PTDSS1 missense mutation within exon 7 (c.829T>C, p. Trp277Arg) near the three previously reported PTDSS1 mutations (Fig. 4). Her defect was veriﬁed by its absence in the SNP database. In fact, we found the Trp277 of PSS1 to be evolutionarily conserved (Fig. 5). A change of tryptophan to arginine would be considered, according to PolyPhen-2
(http://genetics.bwh.harvard.edu/pph2/index.shtml), “probably damaging” with a score of 1.00 on a scale of up to 1.00. The father did not have the mutation (maternal DNA was not available).
Discussion Little is known about the skeletal disease of LMHD apart from its striking clinical presentation and remarkable radiographic features.(1,2) LMHD causes loose skin where elastic tissue is lacking.(14) Enamel dysplasia is characteristic.(6) Intellectual impairment is severe.(1,2) However, there have been no descriptions of bone histopathology or results from assaying BTMs.
Fig. 4. PTDSS1 mutation in our patient. (A) Gene structure of PTDSS1 showing 13 exons and locations of LMHD mutations. Our patient’s mutation is noted with an asterisk. (B) Electropherograms of forward and reverse DNA sequence show our patient’s mutation in exon 7. (C) Control and patient cDNA sequence (as codons) and amino acid sequence show our patient’s defect (the altered nucleotide and amino acid changes are in bold and underlined).
Journal of Bone and Mineral Research
Fig. 5. Evolutionary alignment and cellular location for our patient’s PTDSS1 mutation. (A) Evolutionary alignment of amino acid sequence in the region of PTDSS1 exon 7 containing the mutation, using the alignment tool at the UCSC Genome Browser (https://genome.ucsc.edu). The boxed amino acid, tryptophan (W), is the affected amino acid. It is conserved throughout all species tested, indicating it is an important amino acid in the function of PSS1 protein. Underlined amino acids are those that are not conserved in some species. (B) Proposed PSS1 protein structure showing nine transmembrane domains within a lipid bilayer and the known LMHD mutations. Our patient’s mutation is denoted with an asterisk. “ER Lumen” and “Cytosol” indicate the orientation of PSS1 in the lipid bilayer. The proposed PSS1 structure is based on UniProt data for PSS1 (P48651: www.uniprot.org).
The radiographic features of the LMHD skeleton include delayed closure of the anterior fontanel with progressive osteosclerosis of the skull, including the petrous bones and base as well as the mandible and maxilla.(1,2) The ribs tend to be wide and sclerotic. The clavicles are dense and widened at their medial two-thirds. The entire spine and pelvis are sclerotic. Osteosclerosis affects the extremities, but later some demineralization involves the epiphyses and metaphyses along with diaphyseal undermodeling and cortical thickening.(1,2) The hands and feet are grossly abnormal with short middle and proximal phalanges, symphalangism, syndactyly, and delayed ossiﬁcation of the lateral metatarsals and ulnar-sided metacarpals.(1,2) In 2007, Dateki and colleagues(16) reported that bone scintigraphy of mild LMHD showed increased radiopharmaceutical uptake in sclerotic regions identiﬁed radiographically. In 1974, Kaye and colleagues(14) reported among postmortem ﬁndings at age 54 days “the parietal and occipital bones were undeveloped, and wormian bones were present in the paramedian region.” In 1977, Robinow and colleagues(10) mentioned that although serum ALP was consistently elevated in their patient, calcium, phosphorus, electrolytes, acid phosphatase, and a urinary amino acid chromatogram and metabolic screen were normal. Furthermore, serum PTH was not elevated, and calcitonin was not detectible. In 1983, Gorlin and Whitley(11) noted that “laboratory studies have been unremarkable except for a moderately elevated serum ALP level.” In 2000, Majewski mentioned “low normal” serum ALP.(7) In 2004, Wattanasirichaigoon and colleagues(17) reported normal serum calcium, phosphorus, and ALP levels. Investigation of our patient’s mineral homeostasis disclosed an elevated serum calcium level while she received considerably more calcium from her formula than recommended in the RDA.(18) Concomitantly, her serum intact PTH level was not suppressed and her urine calcium level was not elevated despite the apparent hypercalcemia—perhaps inﬂuenced by her positive mineral balance. Although the apparent presence of a small amount of serum CK-BB in her circulation suggested an osteopetrosis,(19) this
WHYTE ET AL.
CK-BB may have derived from her central nervous system complications. Absence of elevated serum total CK or TRAP5b levels provided evidence against an OC-rich osteopetrosis from compromised OC action.(19,20) In fact, her normal levels of urinary OHP and free DPD indicated that bone resorption was not suppressed, although BTM interpretation is perhaps unclear when skeletal mass is greatly increased. Instead, her BTMs included slightly elevated serum levels of ALP, BAP, and OCN that suggested accelerated bone formation. Exuberant bone formation in LMHD is understandable when there is PTDS excess (see below). In 2014, Sousa and colleagues(3) discovered three heterozygous missense mutations within PTDSS1 (c.1058A>G, p. Gln353Arg; c.805C>T, p.Pro269Ser; and c.794T>C, p.Leu265Pro) among 5 unrelated patients with LMHD. These investigators had studied the three LMHD patients reported by Saraiva,(12) Chrzanowski and colleagues,(15) and Wattanasirichaigoon and colleagues,(17) as well as 2 new LMHD patients. One mutation was within exon 9, and the other two mutations were within exon 7. Our patient’s de novo heterozygous missense mutation (c.829T>C, p.Trp277Arg) was similarly within exon 7 and would change the amino acid tryptophan (Trp) to arginine (Arg), which we found is conserved across all species listed (Fig. 5A). The change is “nonconservative” because tryptophan is highly hydrophobic and uncharged, whereas arginine is hydrophilic and charged. PTDS is produced in mammalian cells by two synthases: 1) PSS1, encoded by PTDSS1, through exchange of L-serine for the choline moiety of phosphatidylcholine; and 2) PSS2, encoded by PTDSS2, through conversion of phosphatidylethanolamine to PTDS by a parallel base-exchange reaction.(21–23) PTDS represents 3% to 10% of all mammalian membrane phospholipid in tissues and in cells in culture,(23) and has considerable importance reﬂecting its unique physical and biochemical properties,(24) as well as its speciﬁc tissue(22,25) and subcellular distributions.(22,26) At cell surfaces, PTDS acts in apoptosis, coagulation, and internalization of viruses.(21) Intracellularly, PTDS interacts with key signaling proteins such as the Ras and
Journal of Bone and Mineral Research
Rho family of GTPases, as well as with protein kinase C that mediates many cellular responses.(22,27) Mice lacking either Ptdss1 or Ptdss2 are phenotypically normal, indicating that these murine genes and products can compensate for one another,(28,29) but simultaneous disruption of both genes is lethal. Thus, PTDS seems necessary for survival.(28) PTDSS1 was mapped in 1994 to chromosome 8 (8q22).(30) In 2014, Sousa and colleagues(3) demonstrated that the PTDSS1 mutations of LMHD profoundly increased PTDS biosynthesis in ﬁbroblasts. However, immunoblotting showed that cellular PSS1 protein was not elevated.(3) Furthermore, no compensatory changes were present in the levels of PTDSS2 mRNA or PSS2 activity that could account for accelerated biosynthesis of PTDS.(3) Therefore, the high rate of PTDS (and phosphatidylethanolamine) synthesis in LMHD ﬁbroblasts was not attributable to increased amounts of PSS1. Notably in these LMHD ﬁbroblasts, [3H]serine incorporation into PTDS and phosphatidylethanolamine was profoundly resistant to inhibition by PTDS,(3) yet the cellular levels of PTDS, phosphatidylethanolamine, and phosphatidylcholine remained normal. Hypothetically, any increase in PTDS synthesis might not increase cellular steady-state PTDS levels because these are tightly regulated.(31) Instead, the phospholipid content of LMHD tissues or subcellular organelles may have been altered and consequently disturbed important signaling processes.(3) PSS1 in the endoplasmic reticulum and mitrochondria has nine transmembrane domains. All four LMHD mutations would disrupt the luminal side of PSS1.(3) LMHD could arise from contextdependent mechanisms and tissue- and subcellular locationspeciﬁc consequences of the increased PTDS synthesis. However, Sousa and colleagues(3) discussed why the mechanism seemed unlikely to be enhanced p53-dependent apoptotic cell death. We identiﬁed an elevated level of phosphoserine in our patient’s urine, perhaps somehow related to the increased PTDS biosynsthesis. Whether hyperphosphoserinuria characterizes LMHD will, however, require investigation of future patients. PTDSS1 is expressed more in brain than bone.(25) Based on a human developmental transcriptome, this expression occurs primarily during the ﬁrst trimester within the forebrain. The head CT reported by Wattanasirichaigoon and colleagues(17) for their patient with relatively mild LMHD, who was later found to have a PTDSS1 mutation,(3) did not show the profound changes documented in our patient. The MRI study reported by Saravia(12) featured hypoplasia mainly of the splenium of the corpus callosum but also of the genu. Thus, PTDSS1 has importance for brain development. Perhaps our patient’s slow recovery from anesthesia was related to excessive levels of PTDS in the lipid bilayer of her neurons, posing a caution for future LMHD patients. Before Sousa and colleagues(3) in 2014, there was no link between PTDS metabolism and an osteosclerotic dysplasia (especially those classiﬁed with LMHD in group 24 of the nosology of genetic skeletal disorders).(32) However, PTDS is known to interact with calcium for bone mineralization, dentine formation, and physiological and pathological calciﬁcation.(4,33) Within matrix vesicles, PTDS binds calcium to initiate hydroxyapatite crystal formation during phase 1 mineralization.(4,34) Notably, osteointegrative biomaterial studies(33,34) involving phospholipids (including phosphatidylcholine, phosphatidylinositol, and PTDS) coated on titanium and complexed with calcium and phosphate, disclosed that only PTDS would enhance protein synthesis and ALP activity.(4,34) PTDS (and PTDS-mimicking) coating technology promotes calciﬁcation and has therapeutic potential including for dental implantology.(35) Hence, PTDS is a functional molecule with promise for bone-repairing biomaterials
Journal of Bone and Mineral Research
and bone tissue engineering.(33,34) Among reports that PTDS promotes bone mineralization,(4,34) Xu and colleagues(5) in 2013 showed that PTDS treatment enhances osteogenic differentiation of human mesenchymal stem cells in culture via ERK-signal pathways to include the osteogenic gene markers ALP, OCN, and RUNX2. Additionally, PTDS-containing liposomes inhibited OC differentiation and prevented trabecular bone loss.(35) Thus, the net effect of PTDS excess in LMHD would be increased bone mass. In this regard, it will be important in future studies to delineate the skeletal histomorphometry of LMHD. In conclusion, our ﬁndings from an infant with LMHD afﬁrm that heterozygous mutation of PTDSS1 can cause LMHD, and our discovery of hyperphosphoserinuria in LMHD (which must be veriﬁed in other LMHD patients) supports a pathogenesis from activation of PTDSS1 leading to enhanced biosynthesis of PTDS and then accelerated bone formation.
Disclosures All authors state that they have no conﬂicts of interest.
Acknowledgments We are grateful to Dr Lyle Johnson and Dr John Fangman (Minneapolis, MN, USA) for referring the patient and for providing medical records. The study was made possible by the skill and dedication of the nursing, laboratory, and dietary staff, Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, MO, USA. Ms Margaret Huskey and Ms Shenghui Duan helped perform the PTDSS1 mutation analysis. Dr Gary S Gottesman provided a description of the patient’s dysmorphology. Ms Sharon McKenzie provided expert secretarial help. This study was funded in part by Shriners Hospitals for Children, The Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund, The Hypophosphatasia Research Fund, The Barnes-Jewish Hospital Foundation, The Frederick S. Upton Foundation, and the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award number DK067145. Amanda Blythe was supported by the STARS (Students and Teachers as Research Scientists) Program from the University of Missouri-St. Louis, St. Louis, MO, USA. The content of the article is solely the responsibility of the authors and does not necessarily represent the ofﬁcial views of the National Institutes of Health. Authors’ roles: MPW coordinated investigation of the patient and drafted and ﬁnalized the manuscript. AB performed the mutation analysis. WHM detailed the patient’s radiologic ﬁndings and interpreted published reports. ARN provided patient care and research chart review. VB illustrated the manuscript and performed literature searches. SM guided the mutational analysis and interpretation of the molecular ﬁndings
References 1. Spranger JW, Brill PW, Poznanski A. Lenz-Majewski hyperosteotic dysplasia. In: Bone dysplasias: an atlas of genetic disorders of skeletal development. 2nd ed. New York: Oxford University Press; 2002; pp 528–31. 2. Online Mendelian Inheritance in Man, OMIM1 [Internet]. Baltimore: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University. Available at: http://omim.org/. Accessed September 30, 2014.
3. Sousa SB, Jenkins D, Chandudet E, et al. Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause LenzMajewski syndrome. Nat Genet. 2014;46:70–6. 4. Wu LNY, Genge BR, Wuthier RE. Analysis and molecular modeling of the formation, structure, and activity of the phosphatidylserinecalcium-phosphate complex associated with biomineralization. J Biol Chem. 2008;283:3827–38. 5. Xu C, Zheng Z, Fang L, et al. Phosphatidylserine enhances osteogenic differentiation in human mesenchymal stem cells via ERK signal pathways. Mater Sci Eng C Mater Biol Appl. 2013;33:1783–8. 6. Lenz WD, Majewski F. A generalized disorder of the connective tissues with progeria, choanal atresia, symphalangism, hypoplasia of dentine and craniodiaphyseal hyperostosis. Birth Defects Orig Art Ser. 1974;10:133–6. 7. Majewski F. Lenz-Majewski hyperostotic dwarﬁsm: reexamination of the original patient. Am J Med Genet. 2000;93:335–8. 8. Macpherson RI. Craniodiaphyseal dysplasia, a disease or group of diseases. J Can Radiol. 1974;25:22. 9. Braham RL. Multiple congenital abnormalities with diaphyseal dysplasia (Camurati-Engelmann’s Syndrome). Oral Surg. 1969;27:20. 10. Robinow M, Johanson AJ, Smith TH. The Lenz-Majewski hyperostotic dwarﬁsm. A syndrome of multiple congenital anomalies, mental retardation, and progressive skeletal sclerosis. J Pediatr. 1977;91: 417–21. 11. Gorlin RJ, Whitley CB. Lenz-Majewski syndrome. Radiology. 1983; 149:129–31. 12. Saraiva JM. Dysgenesis of corpus callosum in Lenz-Majewski hyperostotic dwarﬁsm. Am J Med Genet. 2000;91:198–200. 13. Nishimura G, Harigaya A, Kuwashima M, Kuwashima S. Craniotubular dysplasia with severe postnatal growth retardation, mental retardation, ectodermal dysplasia, and loose skin: Lenz-Majewskilike syndrome. Am J Med Genet. 1997;71:87–92. 14. Kaye CI, Fisher DE, Esterly NB. Cutis laxa, skeletal anomalies, and ambiguous genitalia. Am J Dis Child. 1974;127:115–7. 15. Chrzanowska KH, Fryns JP, Krajewska-Walasek M, Van den Berghe H, Wisniewski L. Skeletal dysplasia syndrome with progeroid appearance, characteristic facial and limb anomalies, multiple synostoses, and distinct skeletal changes: a variant example of the LenzMajewski syndrome. Am J Med Genet. 1989;32:470–4. 16. Dateki S, Kondoh T, Nishimura G, et al. A Japanese patient with a mild Lenz-Majewski syndrome. J Hum Genet. 2007;52:686–9. 17. Wattanasirichaigoon D, Visudtibhan A, Jaovisidha S, Laothamatas J, Chunharas A. Expanding the phenotypic spectrum of Lenz-Majewski syndrome: facial palsy, cleft palate and hydrocephalus. Clin Dysmorphol. 2004;13:137–42. 18. Otten JJ, Hellwig JP, Meyers LD. National Research Council. Dietary reference intakes: the essential guide to nutrient requirements. Washington, DC: The National Academies Press; 2006. 19. Whyte MP, Chines A, Silva DP, Landt Y, Ladenson JH. Creatine kinase brain isoenzyme (BB-CK) presence in serum distinguishes osteopetroses among the sclerosing bone disorders. J Bone Miner Res. 1996;11:1438–43.
WHYTE ET AL.
20. Whyte MP, Kempa LG, McAlister WH, Zhang F, Mumm S, Wenkert D. Elevated serum lactate dehydrogenase isoenzymes and aspartate transaminase distinguish Albers-Schőnberg disease (chloride channel 7 deﬁciency osteopetrosis) among the sclerosing bone disorders. J Bone Miner Res. 2010;25: 2515–26. 21. Sturbois-Balcerzak B, Stone SJ, Sreenivas A, Vance JE. Structure and expression of the murine phosphatidylserine synthase-1 gene. J Biol Chem. 2001;276:8205–12. 22. Vance JE, Tasseva G. Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells. Biochim Biophys Acta. 2013;1831:543–54. 23. Tomohiro S, Kawaguti A, Kawabe Y, Kitada S, Kuge O. Puriﬁcation and characterization of human phosphatidylserine synthases 1 and 2. Biochem J. 2009;418:421–9. 24. Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys. 2010;39:407–27. 25. Mozzi R, Buratta S, Goracci G. Metabolism and functions of phosphatidylserine in mammalian brain. Neurochem Res. 2003;28: 195–14. 26. Schick PK, Kurica KB, Chacko GK. Location of phosphatidylethanolamine and phosphatidylserine in the human platelet plasma membrane. J Clin Invest. 1976;57:1221–6. 27. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992;258:607–14. 28. Arikketh D, Nelson R, Vance JE. Deﬁning the importance of phosphatidylserine synthase-1 (PSS1): unexpected viability of PSS1-deﬁcient mice. J Biol Chem. 2008;283:12888–97. 29. Bergo MO, Gavino BJ, Steenbergen R, et al. Deﬁning the importance of phosphatidylserine synthase 2 in mice. J Biol Chem. 2002;277: 47701–8. 30. Nomura N, Miyajima N, Sazuka T, et al. Prediction of the coding sequences of unidentiﬁed human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1. DNA Res. 1994;1:27–35. Note: Erratum: DNA Res. 1995;2:210 only. 31. Vance DE, Vance JE. Physiological consequences of disruption of mammalian phospholipid biosynthetic genes. J Lipid Res. 2009;50: S132–7. 32. Warman ML, Cormier-Daire V, Hall C, et al. Nosology and classiﬁcation of genetic skeletal disorders: 2010 revision. Am J Med Genet A. 2011;155A:943–68. 33. Merolli A, Santin M. Role of phosphatidyl-serine in bone repair and its technological exploitation. Molecules. 2009;14:5367–81. 34. Satsangi A, Satsangi N, Glover R, Satsangi RK, Ong JL. Osteoblast response to phospholipid modiﬁed titanium surface. Biomaterials. 2003;24:4585–9. 35. Wu Z, Ma HM, Kukita T, Nakanishi Y, Nakanishi H. Phosphatidylserinecontaining liposomes inhibit the differentiation of osteoclasts and trabecular bone loss. J Immunol. 2010;184:3191–201.
Journal of Bone and Mineral Research