2018, 65 (2), 227-238

Note

Definitive diagnosis of mandibular hypoplasia, deafness, progeroid features and (MDPL) syndrome caused by a recurrent de novo in the POLD1

Haruka Sasaki1), 2), Kumiko Yanagi3) *, Satoshi Ugi4), Kunihisa Kobayashi1), Kumiko Ohkubo5), Yuji Tajiri6), Hiroshi Maegawa4), Atsunori Kashiwagi7) and Tadashi Kaname3) *

1) Department of and Mellitus, Fukuoka University Chikushi Hospital, Chikushino, Fukuoka 818-8502, Japan 2) Division of Diabetic Medicine, Bunyukai Hara Hospital, Ohnojo, Fukuoka 816-0943, Japan 3) Department of Genome Medicine, National Research Institute for Child Health, Setagaya, Tokyo 157-8535, Japan 4) Department of Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan 5) Department of Laboratory Medicine, School of Medicine, Fukuoka University, Jonan-, Fukuoka 814-0180, Japan 6) Division of Endocrinology and Metabolism, Kurume University School of Medicine, Kurume, Fukuoka 830-0111, Japan 7) Diabetes Center, Seikokai Kusatsu General Hospital, Kusatsu, Shiga 525-8585, Japan

Abstract. Segmental with lipodystrophy are extremely rare, heterogeneous, and complex multi-system disorders that are characterized by phenotypic features of premature aging affecting various tissues and organs. In this study, we present a “sporadic/isolated” Japanese woman who was ultimately diagnosed with mandibular hypoplasia, deafness, progeroid features, and progressive lipodystrophy (MDPL) syndrome (MIM #615381) using whole exome sequencing analysis. She had been suspected as having atypical and/or progeroid syndrome based on observations spanning a 30-year period; however, repeated genetic testing by Sanger sequencing did not identify any causative mutation related to various subtypes of congenital partial lipodystrophy (CPLD) and/or mandibular dysplasia with lipodystrophy (MAD). Recently, MDPL syndrome has been described as a new entity showing progressive lipodystrophy. Furthermore, polymerase delta 1 (POLD1) gene on chromosome 19 have been identified in patients with MDPL syndrome. To date, 21 cases with POLD1-related MDPL syndrome have been reported worldwide, albeit almost entirely of European origin. Here, we identified a de novo mutation in 15 (p.Ser605del) of the POLD1 gene in a Japanese case by whole exome sequencing. To the best of our knowledge, this is the first identified case of MDPL syndrome in Japan. Our results provide further evidence that mutations in POLD1 are responsible for MDPL syndrome and serve as a common genetic determinant across different ethnicities.

Key words: Lipodystrophy, Mandibular hypoplasia, Progeroid features, POLD1 mutation, Japanese case

CONGENITAL comprise a severe insulin resistance, glucose intolerance, diabetes, very rare group of heterogeneous disorders that affect dyslipidemia, and fatty liver disease [1-6]. Recently, the adipose tissue distributions and are characterized by genetic origins of some cases have been clarified. A pro‐ varying degrees of body fat loss (complete and/or partial) totypic example is Werner syndrome, which is a rare and various profound metabolic derangements such as autosomal recessive syndrome (MIM #277700) charac‐ terized by a variety of clinical signs of premature aging Submitted Jul. 12, 2017; Accepted Oct. 23, 2017 as EJ17-0287 that has been shown to be caused by biallelic germline Released online in J-STAGE as advance publication Dec. 2, 2017 mutations in the Werner gene (WRN: encoding a Correspondence to: Haruka Sasaki, MD, PhD., Fukuoka Univer‐ RecQ DNA helicase/) [7]. Mandibular dys‐ sity, 1-2-6-1202, Momochihama, Sawara-ku, Fukuoka 814-0001, Japan. plasia associated with lipodystrophy (MAD) type A E-mail: [email protected] (MIM #248370) and type B (MIM #608612) comprise *These authors equally contributed to this work. another type of rare autosomal recessive syndrome,

©The Japan Endocrine Society 228 Sasaki et al.

Table 1 Classification of genetic subtypes of lipodystrophy Clinical subtype/Eponym Gene Chromosome Locus Inheritance transmission OMIM Generalized CGLD1 AGPAT2 11q13 AR #608594 CGLD2 BSCL2 9q34.3 AR #269700 CGLD3 CAV1 11q13 AR #612526 CGLD4 PTRF 17q21.2 AR #613327 MAD-A type LMNA 1q22 AR #248370 Partial CPLD1/Köbberling variety NK NK AD #608600 CPLD2/Dunnigan variety LMNA 1q22 AD #151660 CPLD3 PPARγ 3p25.2 AD #604367 CPLD4 PLIN1 15q26.1 AD #613877 CPLD5 CIDEC 3p25.3 AR #615238 CPLD6 LIPE 19q13.2 AD #615980 CPLD7 AKT2 19q13.3 AD #125853 CPLD8 ADRA2A 10q25.2 AD #104210 MAD-B type ZMPSTE24 1q34.2 AR #608612 HGPS LMNA 1q22 Heterozygous/de novo #176670 SHORT syndrome PIK3R1 5q13.1 AD #269880 Werner syndrome WRN 8p12 AR #277700 Nestor-Gullermo syndrome BANF1 11q13.1 AR #614008 PRAAS/JMP PMMB8 6p21.32 AR #256040 MDPL syndrome POLD1 19q13.33 Heterozygous/de novo #615381 CGLD, congenital generalized lipodystrophy; CPLD, congenital partial lipodystrophy; MAD, mandibuloacral dysplasia; MDPL, mandibular hypoplasia, deafness, progeroid features, lipodystrophy; HGPS, Hutchinson-Gilford Progeria syndrome; PRSSA, - associated autoinflammatory syndrome; JMP, joint contractures, muscle , microcytic anemia and panniculitis-induced lipodystrophy; AD, autosomal dominant; AR, autosomal recessive; OMIM, Online of Mendelian Inheritance in Man; NK, not known

resulting in lipodystrophy and progeroid features caused disease through repeated Sanger sequencing [10]. by mutations of the A/C gene (LMNA) in type A Notably, a new causal factor of progressive lipodystro‐ and the zinc metalloprotease (ZNPTSTE24) gene in type phy associated with MDPL syndrome has been identified B [8, 9]. However, many others remain to be elucidated [11-17]. Mutations in the polymerase delta 1 (POLD1) [1-4]. Currently, the comprehensive definition and classi‐ gene have been detected in several patients with MDPL, fication of lipodystrophy is a work in progress with up to suggesting that MDPL is the result of POLD1 gene 20 loci described as being associated with infrequent li‐ mutation. Furthermore, the reported clinical features of podystrophy syndromes [2, 4, 5] (Table 1). MDPL possessing POLD1 gene mutations were very Recently, we reported a case of congenital partial lipo‐ similar to and overlapped with those of our case. This dystrophy with mandibular hypoplasia, deafness, and prompted us to question whether our case in fact repre‐ progeroid features (MDPL) syndrome as a novel subtype sented MDPL syndrome caused by a POLD1 gene muta‐ of lipodystrophy, distinct from known types of congeni‐ tion, as no mutation had yet been detected. Therefore, in tal partial lipodystrophy (CPLD) and/or MAD, because the current study we investigated the complete POLD1 we could not detect any gene mutations related to such a gene in our patient along with her parents and elder MDPL syndrome caused by a POLD1 mutation 229 brother using whole exome sequencing analysis. In addi‐ infusion was 167 mg/dL (RV: 73 ± 13.4) [18]. Antibod‐ tion, we summarized 21 previously reported cases with ies to insulin or the insulin receptor as well as mitochon‐ MDPL syndrome in the literature and compared them drial DNA mutations (A3243G, A8296G, A8344G, with classical Werner syndrome. T8356C G8361A, and G8363A) were all negative. She was suspected to have “atypical progeroid syndrome Case Description (Werner syndrome)” [19, 20], although no definitive diagnosis was obtained. There was no family history of A Japanese female (currently more than 46 years old) any similar disorders (Fig. 1). When she was 33 years was born at full term to non-consanguineous parents with old (Wt 30.8 kg, Ht 142.7 cm, body mass index [BMI] a normal birth appearance (weight [Wt] 3.65 kg, height 15.1 kg/m2), she presented with an overall paucity of [Ht] 49.5 cm). Her parents first noted her muscle spasms subcutaneous fat (total fat mass 14.9% by bioimped‐ and fat loss in her limbs during later infancy, and she ance). She had prominent with conjunctival telean‐ subsequently developed poorly overall. Bilateral sensori‐ giectasia, but no juvenile . Her menarche had neural hearing loss was detected, and she required hear‐ occurred at age 13 followed by regular cycles for five ing aids before . At 14 years of age, she was years. In addition, she showed mild hirsutism in the admitted to the university hospital for her short stature lower extremities and acanthosis nigricans in the axillae (Wt 28.6 kg, Ht 139.2 cm; both <3.0 SD, despite hyper‐ and neck regions with alopecia aerate of the scalp. She phagia) and frequent muscle cramps. Subcutaneous fat developed oligo-/amenorrhea owing to polycystic ovary loss was noted predominantly on her facial cheeks and syndrome (POCS) with abnormal sex hormone levels, extremities with pigmentation over a wide area. An oral including low sex hormone binding globulin (SHBG), glucose tolerance test (O-GTT: 1.7 g glucose/kg) showed elevated luteinizing hormone (LH)/follicular-stimulating normal glucose tolerance with increased insulin resis‐ hormone (FSH) ratio on GnRH stimulation test, and a tance (homeostatic model assessment-insulin resistance multi-cystic pattern on magnetic resonance imaging (MRI). [HOMA-IR] score of 5.3, reference value [RV]: <1.6; Furthermore, both abdominal computed tomography fasting plasma glucose [FPG] and immunoreactive insu‐ (CT) and MRI confirmed marked visceral fat accumula‐ lin [F-IRI] of 74 mg/dL and 29 μU/mL, respectively). An tion (visceral and subcutaneous fat areas [VFA/SFA] on endocrine investigation including evaluation of growth CT were 217.1/13.3 cm2 at the navel level, respectively) hormone [GH; L-DOPA and arginine provocative test] with mandibular hypoplasia and torus mandibularis and insulin-like growth factor 1 (IGF-1) levels, thyroid without acro-osteolysis and clavicular hypoplasia. Low hormone and adrenal function, and computed tomogra‐ plasma leptin/adiponectin levels were observed (3.6 phy (CT) of the head including the pituitary showed no ng/mL and 2.4 μg/mL, respectively, RV: 7.4 ± 3.7 and abnormalities. Findings of electroencephalography, nerve 11.7 ± 1.0 for females). The 75-g O-GTT revealed a dia‐ conduction, and electromyography were also normal. betic pattern (FPG and 2 h-glucose levels were 103 and She had a normal karyotype (46, XX). 230 mg/dL) with hyperinsulinemia (F-IRI and 2 h-IRI At 21 years of age, biopsy-confirmed non-alcoholic levels were 23.8 and 226.9 μU/mL), respectively. Severe steatohepatitis (aspartate aminotransferase [AST] 151, insulin resistance was confirmed by the glucose clamp alanine aminotransferase [ALT] 262, and gamma-glutamyl technique (glucose infusion rate 4.32 mg/kg/min at an transferase [γGTP] 163 IU/L, respectively) with dyslip‐ insulin infusion rate of 1.5 mU/kg/min, RV: 10.9 ± 4.2). idemia (total cholesterol [TC] 258, high density lipopro‐ She was hypertensive with a blood pressure of 175/86 tein [HDL] cholesterol 33, and triglycerides [TG] 458 mmHg, and Doppler ultrasonography showed evidence mg/dL), impaired glucose tolerance (IGT), increased gly‐ of intima-media thickness (IMT: max 1.6 mm, RV: <0.6 cated hemoglobin (HbA1c: NGSP 5.7%), and marked mm). Pioglitazone with telmisartan was prescribed con‐ endogenous hyperinsulinemia (FPG 82 mg/dL, F-IRI 30 tinuously from 33 to 44 years of age (i.e., approximately μU/mL, and fasting C-peptide, 5.9 ng/mL) were ob‐ 10 years). These treatments resulted in significantly served. Progeroid features, including a prematurely aged improved metabolic parameters such as hyperglycemia, face, high-pitched voice, and atrophic over a wide hyperinsulinemia, plasma hypo-leptin/adiponectinemia, area and a small lip with a small mandible (jaw) were menstrual irregularity and hyperphagia. However, there also noted. The value of steady state plasma glucose were no satisfactory changes in the affected lipoatrophic (SSPG) assessed using somatostatin, glucose, and insulin regions, and a further increase in visceral fat (VFA/SFA; 230 Sasaki et al.

Fig. 1 Pedigree of the patient’s family. Family members all show relatively long survival.

301.5/20.4 cm2 at the age 45 years). bio-bwa.sourceforge.net/). Variants were called using the To establish the genetic causes, a sequence analysis GATK Unified Genotyper and using ANNOVA (http:// was repeatedly performed during the follow-up period; annover.openbioinfomatics.org/en/latest/). A detected however, no key mutations in associated with vari‐ POLD1 variant was validated by Sanger sequencing. Pri‐ ous types of CPLD and/or MAD A/B types including mers 5'-TAAAGGGTGAGGCCACAAGA-3' and 5'- LaminA/C, ZMPSTE24, and others (i.e., LMNB2, CCCTCCACTCCCTACTATCTCTG-3' were used for PPARG, insulin receptor, IRS-1, Cav-1, Cav-2, BSCL2, direct sequencing of exon 15 of the POLD1 gene. AGPAT2, CICDEC, PTRF, AKT-2, and PLIN-1 were identified [1-4] (Table 1). Therefore, at that time we Results described this case as a novel subtype of CPLD and/or a genetic syndrome of unknown cause [10]. We sequenced 7,903,970,560 bp in total in the patient. The average throughput in terms of the depth of whole Materials and Methods sequences was approximately 120x. After exclusion of common variants, 18,610 single nucleotide variants The patient and her family members gave written (SNVs; including indels) were called. Among them informed consent for genetic testing and the study was 1,102 SNVs were nonsynonymous or splice site variants. approved by the National Research Institute for Child After filtering of variants between the patient and her Health and Development, Initiative on Rare and Undiag‐ parents, we identified a de novo three-base nosed Disease in Pediatrics (IRUD-P), Japan [21]. (NM_001256849_1814CTC, p.Ser605del) in exon 15 of Genomic DNA was extracted from the peripheral the POLD1 gene. Because the read count of the deletion blood of the patient, her parents, and her brother using a was approximately 50% at the variant site, the variant QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany). was heterozygous but not mosaic in the patient (Fig. 2A, We performed whole-exome sequencing (WES) analysis B). The deletion was located on the C-terminal side of using the SureSelect Human All Exon V6 kit (Agilent the DNA polymerase domain and predicted as disease- Technology, Santa Clara, CA, USA) for capture and a causing by Mutation Taster. The variant was not found HiSeq2500 (Illumina, San Diego, CA, USA) for se‐ in the 1000 Genome database (http://www.international quencing with 101-bp paired-end reads. Reads were genome.org/), dbSNP (http://www.nebi.nlm.gov/SNP), aligned to CRC37 using Burrows-Wheeler Aligner (http:// genome Aggregation Database (genome AT); http:// MDPL syndrome caused by a POLD1 mutation 231

Fig. 2 Identification of the POLD1 mutation and protein structure of POLD1. The position of the mutation in the patient’s gene is marked with a vertical arrow (A). The mutation is located in the polymerase active site. Both parents and the patient’s brother were negative for the variant. Whole exome sequencing IGV browser images of the POLD1 exon 15 region and read counts of the wild type and deletion (WT, del) in each individual (B). Sanger sequencing of parts of POLD1 exon 15. The father, mother, and brother showed the wild-type (WT) sequence. Arrow indicates the frame shift start (C). gnomad.broadinstitute.org/), Japanese genome variant features, and lipodystrophy syndrome (MDPL syndrome) database [22] or in-house data for exome sequencing. [11-17]. In addition, no pathogenic mutations were also The variant was confirmed by Sanger sequencing and found in other lipodystrophy-related genes, including was not found in her parents and healthy brother, sup‐ FBN1, LIPE, NK, PIK3R1, PSMB8, RTRF, and BANF1, porting that the variant was de novo and pathogenic (Fig. as well as in the Werner helicase gene (WRN) by either 2C). The deletion was reported as a pathogenic variant in Sanger sequencing or WES [21]. patients with mandibular hypoplasia, deafness, progeroid Thus, according to both the unique phenotypic mani‐ 232 Sasaki et al.

Table 2 Summary of ethnicity, mutations, and sex differences [11-17 and this case] Mutation Sex Age at referral† Ethnicity p.Ser605del p.Arg507Cys p.Ile1070Asn p.Glu1067Lys Male/Female Children/Adults Italian 3 2 / / 0/5 0/5 French 3* / / / 1/2 1/2 British 3 / 1 1/3 1/3 US, mixed European 1 1 / / 1/1 0/2 Swedish 1 / / / 1/0 0/1 Hungarian 1 / / / 1/0 0/1 Columbian 1 / / / 0/1 1/0 White-Hispanic / / / 2** 0/2 1/1 Indian 1 / / / 1/0 0/1 Chinese 1 / / / 1/0 1/0 Japanese (our case) 1 / / / 0/1 1/0 Total 16 3 1 2 7/15 6/16

/: None, † Children (≦16 yrs old), * Mother and son, ** Mother and daughter festations and the WES analysis, the patient was diag‐ and progeroid features featuring subcutaneous fat loss nosed as having MDPL syndrome; MIM #615381 in the and severe insulin resistance (MDPL syndrome). They Online Mendelian Inheritance in Man database [11-17]. identified an in-frame heterozygous deletion at the polymerase-active site in exon 15 of POLD1, c.1812 Discussion 1814delCTC (p.Ser605del) as the cause of the MDPL syndrome [11]. Subsequently, a heterozygous missense We identified a mutation of the POLD1 gene in a Jap‐ mutation (c1519>T, p.Arg507Cys: substitution of an anese patient who we had initially diagnosed as having a arginine at codon 507 with a cysteine) in exon 13 of novel subtype of CPLD [10]. Her phenotypic features POLD1 was identified in one Italian patient with MDPL excluding MAD [8, 9] did not fit any of those of CPLD syndrome [13]. Recently, Lessel et al. [14] described [1, 3] such as unique adipose tissue topography, or the POLD1 germline mutations in eight patients of various criteria for classical Werner syndrome, including the car‐ European and South American origins from the dinal signs and symptoms of juvenile cataracts, Achilles International Registry of Werner syndrome (www. tendon calcification, and bone deformation [23-25]. wernersyndrome.org); these patients had been initially To date, 21 patients with POLD1-related MDPL syn‐ diagnosed as having Werner syndrome (Table 3) [23, 24]. drome [11-17, 26] have been reported in English publica‐ More recently, Chen et al. [15] reported a 10-year-old tions, most frequently from European countries (Table Chinese boy with short stature and as the 2). The male-to-female ratio is 7:15. Among them, 15 first MDPL case in Eastern Asia. Additionally, Elouej et patients had p.Ser605del, including our case; three al. [16] very recently described an isolated British female patients had p.Arg507Cys [11-16] and only one had the and a male with MDPL syndrome who had a different novel mutation p.Ile1070Ans [16], and two related POLD1 domains with a new causative mutation (NM female patients had the recently identified mutation, 002691.3:c,3209>A, p.Ile1070Asn in the Zinc Finger 2 p.Glu1067Lys [17]. Thus, the mutation detected in our [ZNF2] domain), which was identified along with a patient, p.Ser605del, might constitute a hotspot for recurrent de novo in-frame deletion p.Ser607del. Further‐ MDPL syndrome (Table 2, Fig. 2). more, Ajluni et al. [17] described two related White- Recently, Weedon et al. [11] described six cases, two Hispanic female patients (mother and daughter) carrying of which were previously reported by Shastry et al. [26], a heterozygous, likely pathogenic novel variant of as a novel syndrome of mandibular hypoplasia, deafness POLD1 (NM_002691.3:c.3199G>A; p.Glu1067Lys) MDPL syndrome caused by a POLD1 mutation 233 from a cross-sectional cohort trial. Thus, our patient may pituitary on MRI. Numerous genomic syndromes associ‐ constitute first report of this syndrome from Japan (Table ated with short stature are caused by genetic defects in 2). All reported POLD1 mutations in patients with fundamental cellular processes [34, 35]. MDPL syndrome are heterozygous, suggesting that this Most of these patients were diagnosed as having syndrome is caused by haploinsufficiency or dominant diabetes or IGT at various ages with insulin resistance negative effect. [12, 16]. The compensatory hyperinsulinemia required to The POLD1 gene belongs to the protein family of maintain glucose homeostasis is associated with a cluster human DNA polymerase delta and encodes the p125 of metabolic derangements that present even before the catalytic subunit. It plays a critical role in genome main‐ onset of diabetes [35]. Hearing impairments constitute tenance through its involvement in replicative DNA syn‐ the most common features in MDPL syndrome, although thesis and multiple synthetic repair processes [27, 28]. they are not always found [14, 15, 17]. The POLD1 gene POLD1 cooperates with WRN, which a gene that is typi‐ is expressed in all cells; therefore, the associated deaf‐ cally associated with Werner syndrome [25, 29]. More‐ ness may be a consequence of axonal neurons not func‐ over, the age-related decrease in POLD1 expression has tioning efficiently [27]. Among the female patients, only been shown to be involved in the DNA repair pathway in three patients had primary/secondary amenorrhea includ‐ vitro [29-31]. The mutation encoding p.Ser605del exists ing our case [10, 13, 17], whereas the others reported a in a highly conserved region of the polymerase δ cata‐ regular menstrual cycle. Insulin is thought to stimulate lytic subunit, and leads to a failure to catalyze DNA po‐ ovarian thecal cells or stromal cells to produce androgen lymerization [11, 27]. In particular, a recombinant Ser605 and decrease the production of SHBG by the liver, deletion mutant exhibited loss of polymerase activity, but resulting in a hyperandrogenism [35-37]. Therefore, the showed little loss of 3' to 5' exonuclease activity [11]. characteristics of our patient support the view that the Therefore, it is speculated that germline mutations in the primary role of sustained hyperinsulinemia may be as a active site of the polymerase might increase replication cause of secondary PCOS, acanthosis nigricans, and stress, resulting in a progeroid disorder [11]. The charac‐ hirsutism [35, 38, 39]. In contrast, undescended testes teristic topography in our case is quite different from that and hypogonadism have been reported in males [14, 16, of MAD A/B type and/or other different genetic diseases 26]. with lipodystrophy syndrome including the Köbberling/ In terms of possible predisposition to Dunnigan variety [1-4] and those termed and , our case only showed mild premature macro‐ [32]. Although roles of some of the affected genes vascular diseases. Notably, our comparative analysis (PPARγ, PLIN1, CIDEC, and AKT2) have been estab‐ (Table 3) suggested that premature atherosclerosis with lished in adipose tissue differentiation or function, hypertension, cardiovascular diseases, and neoplasm insulin signaling and lipid metabolism, the molecular might be less prevalent in MDPL compared with Werner mechanisms underlying selective fat loss remain to be syndrome [24] and other laminopathies [32]. Several resolved. MDPL syndrome and MAD belong to similar germline mutations in POLD1 exonuclease domains are groups as syndromes characterized by premature aging also known to predispose carriers to , particularly and inheritable envelope and/or DNA repair defects [2, colorectal and endometrial cancers [14, 27]. However, to 4, 25]. MDPL syndrome shows a spectrum of clinical date at the age of 46 years, the patient has not presented features that have overlapping manifestations and vari‐ any malignant disease. Although specific muscle in‐ able expression in the development stage of the pheno‐ volvement is typically not reported, most patients do type as shown in Table 3. exhibit muscle atrophy, wasting, and muscle/joint con‐ Mandibular hypoplasia is the most common clinical tractions. Our case showed neither muscle inflammation feature, but is not present in all cases [14]. Additionally, nor degeneration and atrophy on histological examina‐ there is no description of mandibular hypoplasia with tion, and no abnormalities were noted in both electro‐ torus palatinus [33]. Relatively short stature and/or a myography and nerve conduction studies conducted at delayed growth spurt are common in MDPL syndrome. the age of 10 years. Post puberty, these symptoms disap‐ From later infancy, our patient showed poor development peared after prescription of carmazepine. [10] without abnormalities of the GH/IGF-1 axis and Elouej et al. [16] suggested that patients carrying pituitary gland. However, the patient described by Chen p.Ile1070Asn have more severe phenotypic features et al. [15] showed quite low IGF-1 levels and an atrophic associated with repeated respiratory infection than those 234 Sasaki et al.

Table 3 Clinical characteristics and metabolic parameters of our patient compared with 21 previously reported genetically proven patients with POLD1 mutations [11-17] and classical Werner syndrome [24, 25]

*Presented case MDPL (reported 21 cases) †Werner syndrome (%)

14 (at referral), Age (yrs) 6–62# (total 21 cases) 60 s (62%), 50 s (22.7%) 45 (currently) Sex (Male/Female) Female 7/14 Male/Female M82/F93, NA18, (Total: 198) 15 European, 2 US, mixed European, Japanese, Sardinian, Indian/Pakistani, Ethnicity Japanese 2 White-Hispanic, 2 Asian Moroccan Turkish and Dutch Parental consanguinity N 4/4 N, (NA 17) Y (35.3)/Y, affected siblings (kg) 3.65 2.4– 4.2#, (NA 9) NA/low birth weight Normal birth appearance Y 2/2 Y, (NA 19) NA Height (cm) 142.7 114.4 (6 yrs)–191 (23 yrs)# 6 Children (<16 yrs) NA/M:F; 158/148 cm, mean Weight (kg, at yrs) 30.8 12.6 (24 yrs)–60 (62 yrs)# 6 Children (<16 yrs) NA/M:F; 45.3/37, 7kg, mean BMI (kg/m2, yrs) 15.1 8.75 (24 yrs)–26.8 (62 yrs)# NA Age at onset (yrs) 5.5 Infancy-Prepuberty 2.5–10#, (NA 13) NA/over 10 yrs Clinical characteristics Lipodystrophy Y 18/21 Y, 1/21 N NA/Y, regional SC atrophy Whole body fat (%) 14.9 (Impedance, 21yrs) 14.0–24.5#, (NA 13) NA Lack of SC fat loss Y 19/19 Y, (NA 2) Y/loss of SC fat, thin limbs Increased abdominal fat Y 17/18 Y, 1/18 N, (NA 3) NA/truncal obesity Metabolic profiles Normal (14 yrs), 9/21 DM, 2/21 IGT, 9/21 Normal, 2 IR, Y (55.8: DM)/Y Glucose metabolism IGT (21 yrs), DM (33 yrs) 1 DKA, 1 Diet, 1 BG, 2 TZDs, 4 Insulin Y (22.7: IGT)/NA Hyperinsulinemia Y 7/11 Y, (NA 8) Y (IR) F-IRI (μU/mL) 23.8 23.5–58.6 #, (NA 10) NA Glycosylated Hb (HbA1c: %) 5.6 3.3–11.5#, (NA 10) NA Dyslipidemia Y 13/16 Y, 3/16 N, (NA 5) Y/Y, dyslipidemia High TC (mg/dL) Y, 227 64.9–216#, (NA 5) Y (85.4)/NA 13/16 Y, 3/16 N, (NA 3), 174–7004# High triglyceride (mg/dL) Y, 153 Y (59.3)/NA 2 Recurrent pancreatitis High HDL-C (mg/dL) N, 50 3/6 Y, 3/6 N, (NA 15), 27–47# Y (17.2)/NA Leptin (ng/mL)** 3.6 4.4–11.5#, (NA 15) NA Adiponectin (μg/mL)** 2.4 (NA 21) NA Hepatic steatosis Y, confirmed 21 yrs 10/15 Y, 5/15N, (NA 6) Y (50.0)/NA Clinical features Progeroid faces Y (93.3)/Y, pinched faces, Y 19/19 Y, 2/19N (bird-like face, beaked nose) bird-like faces Atrophic skin Y, atopic dermatitis 18/18 Y, (NA 3) Y (93.2)/Y (96) (tight or -like skin) Skin ulcer Y, buttocks 30 yrs (NA 21) Y (86.3)/Y, intractable ulcer Vitiligo of finger Y (NA 21) NA Skin teleangioectasis Y, conjunctival 13/17 Y, 4/17 N, (NA 4), 1 Conjunctival and lid NA, NA Clavus or callus Y (feet and sole) (NA 21) Y (90.4)/Y Flat foot Y 1/1 Y, (NA 20) Y (94.6)/NA Mandibular hypoplasia Y 19/20 Y, 2/20 N, (NA 1) NA Hearing impairment (age of onset, yrs) Y (8–9 yrs) 15/21 Y (4–25 yrs), 6/21 N, 1 lateral deafness NA Voice changes Y 13/17Y, 4/17 N, (NA 4) Y (82.2)/Y, abnormal voice (high pitched/hoarse voice) MDPL syndrome caused by a POLD1 mutation 235

Table 3 Continued

*Presented case MDPL (reported 21 cases) †Werner syndrome (%)

Short stature or Delayed growth Y/Y (95), lack of growth spurt Y 10/13 Y, 6/13 N, (NA 5), 2 rhGH replacement spurt or low birth weight Minimum/maximum (cm) 120 (min.)–191 (max.) Dental crowded (irregular teeth) Y 15/19 Y, 3/19 N, (NA 2) NA Y (97.8)/Y (99), bilateral & juvenile Ocular cataracts N 3/3 N, (NA 18), 2 diabetic retinopathy Prominent Y 3/4 Y, 1/4 N, (NA 17) NA Y/Y (100%, premature graying/ Alopecia (, baldness) Y, alopecia areata 3/3 N, (NA 17), 3 premature graying hair thinning scalp hair Acanthosis nigricans Y (14 yrs) 1/2 Y, 1/2 N, (NA 19) NA (age of recognition, yrs) Hyperthricosis Y (21 yrs) 1/7 Y, 6/7 N, (NA 14) NA Muscle waiting Y 8/10 Y, 2/10 N, (NA 11) NA Muscle contracture or Y 8/11 Y, 3/11 N, (NA 10) NA Joint contracture Y (at 44 yrs)***, Y (60.7)/Y, distal limb bone, (Osteopenia) 0.864 g/cm2 (Lumbar), 6/15 Y, 9/15 N, (NA 6) osteosclerosis distal phalanges 0.561 g/cm2 (Femur) Kyphosis/scoliosis N 6/9 Y, 3/9 N, (NA 12) NA Y (76.5)/Y, extensive soft-tissue Calcification (Achilles tendon) N (X-ray) (NA 19) calcification Menstrual cycle Menarche 13 yrs Oligo/ Menarche 11–15 yrs, (NA 4) 8 Regular periods, 4 NA Loss of (menarche at years) amenorrhea, PCOS Primary/secondary amenorrhea, 1 PCOS 4 Undescended testes, 1 Cryptorchidism & Male hypogonadism / Y/Y, testicular trophy phymosis, 3 Testosterone therapy 4/5 Y, 2/5 N, (NA 14), 1 Proven vertical Children N (unmarried) transmission, 1 Mother & son, 1 Mother & Y/30–40% had children daughter Y (44.4)/Y, cause of death, Neoplasm (Cancer) N (NA 21), 1 Mother died ovarian cancer Non-epithelial tumors etc. 2 Y, 1 death causing respiratory failure (25 yrs), Pulmonary infection N NA, (NA 18) (NA 19) Premature atherosclerosis Y 1/1 Y, (NA 20) Y (25.8)/Y Cerebral hemorrhage N (NA 21) Y (2.5)/NA Cerebral infarction N (NA 21) Y(2.4)/NA Coronary heart disease N 1/1 Y (AMI), (NA 20) Y (11.1)/Y, cause of death obliterans N 1/1 Y, (NA 20) Y (21.6)/NA Hypertension Y 1/1 Y, (NA 20) Y (30.9)/Y, not common Cognitive dysfunctions N 13/15 N, (NA 6) N, adequate for age /seldom Mental retardation N 17/17 N, (NA 4) NA Psychiatric status Depressive state at puberty 2 Anxio-depressive, 1 Bipolar disorder NA 16 p.Ser605del, 2 p.Arg507Cys, 1 p.Ile1070Asn, POLD1 mutation p.Ser605del WRN RecQ DNA helicase 2 p.Glu1067Lys

* Basically described data at 33 yrs old during period of no medication. Y, yes; N, not (absence); NA, not available/no description; F-IRI, fasting immunoreactive insulin; HbA1c, glycated hemoglobin A1c; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; DM, diabetes mellitus; IGT, impaired glucose tolerance; IR, insulin resistance; BG, biguanide; TZDs, thiazolidinediones; rhGH, recombinant human growth hormone; PCOS, polycystic ovary syndrome; DAK, diabetic ketoacidosis; AMI, acute myocardial infarction; WRN, Werner helicase gene. ** Leptin/Adiponectin: Linco Res Ins/Adiponectin ELISA *** Bone mineral density, # range of reported values † Genetically confirmed Werner syndrome (WS: n=47), Takemoto et al. (2013, Japanese WS Working Committee, Japan [24]) and Oshima et al. (2016, International Registry of WS, USA [25]) 236 Sasaki et al. with p.Arg507Cys, whereas the boy with p.Ser605del for diagnosis. This is the first case report of MDPL syn‐ described by Chen et al. [15] was also susceptible to drome in Japan. repeated respiratory infections after infancy. Mutations in POLD1 have been linked to diverse disorders termed Acknowledgements MDPL syndrome; these disorders display heterogeneous phenotypes among individuals and involve diseases We would like to offer our thanks to the patient and including a rare progeroid syndrome [11-17]. Further her family for their collaboration; to Professor Toshifumi accumulation of cases and more cell biological studies Sakata, Fukuoka University Hospital for the long-term are required to clarify the variable spectra of clinical fea‐ follow-up of auditory examinations; and to Editage, tures, multi-systemic involvement, and genotype/pheno‐ Tokyo for English correction. type correlations. It is unclear whether these genetic abnormal findings Disclosure represent only specific/characteristic features at present. Furthermore, the natural history of MDPL syndrome None of the authors have any potential conflicts of remains almost completely unknown, although the long- interest associated with this research. term clinical course of MDPL with diabetes mellitus and hypertension has been well controlled in our case to date, Funding which should encourage patients with similar syndromes. The results suggest that alterations in the DNA replica‐ This work was supported in part by grants from Re‐ tion and/or repair function of POLD1 should be consid‐ search on Intractable Disease (15ek0109034h0002; TK) ered as primary causes of lipodystrophy owing to and the Initiative on Rare and Undiagnosed Disease in adipocytokine dysregulation, mild inflammatory pro‐ Pediatrics (16ek0109166h0002; TK) from the Japanese cesses with impaired adipogenesis, and subsequently the Agency for Medical and Development (AMED). We did development of insulin resistance [40, 41]. Although the not receive any other specific grants from funding agen‐ molecular pathogenesis remains to be fully clarified, our cies in the public commercial or non-profit sectors. results highlight that POLD1 gene mutations are a com‐ mon cause of MDPL syndrome across various ethnicities Authors’ contributions (Table 2). In conclusion, in the present study, we identified a H.S., K.K., and Y.T. were involved in the clinical care genetic mutation of the POLD1 gene in exon 15 and management of the patient during the follow-up (p.Ser605del) that resulted in lipodystrophy in a Japanese period, and contributed to the discussion. H.S., S.U., and woman, identified by sequence analysis of the entire K.K. drafted this manuscript. K.Y., K.O., and T.K. per‐ coding region of the POLD1 gene. An overview of cur‐ formed the WES analysis, including other mutational rent cases of MDPL syndrome suggests a lack of ethnic analyses. Y.T. performed the glucose clamp study and and geographical differences, and the potential for previ‐ contributed to the discussion. S.U., H.M., and A.K. per‐ ous linkage to diverse disorders. Although this syndrome formed the insulin signaling analysis (data not shown) is a fairly rare disease, differential diagnosis through and contributed to the discussion. All authors have comprehensive evaluation of medical history and imag‐ approved the final version of the manuscript. ing findings as well as molecular analysis are essential

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