Partial lipodystrophy, severe dyslipidaemia and insulin resistant diabetes as early signs of Werner syndrome

Abstract

•Partial lipodystrophy is an unusual presentation of Werner syndrome.•Exome analysis revealed a novel splice site variant c.2732+5G>A.•c.2732+5G>A leads to exon skipping and partially abrogates the WRN helicase domain.•The WRN gene should be included in different gene panels to prompt diagnosis. Werner syndrome is a premature ageing disorder caused by biallelic variants in the WRN gene. WRN encodes a dual DNA helicase/exonuclease enzyme. Molecular diagnosis is commonly only made at a late disease stage in the third or fourth decade, when cardinal features have become apparent. We describe a 28 year-old woman who presented with early onset diabetes associated with partial lipodystrophy, severe dyslipidaemia and rapidly progressive liver fibrosis related to non-alcoholic steatohepatitis in the absence of progeroid features. Werner syndrome was diagnosed by trio exome analysis, which revealed compound heterozygous WRN mutations: the known variant c.1290_1293del (p.Asn430Lysfs*7) and the novel intronic splice site variant c.2732+5G>A. cDNA analysis demonstrated this to lead to in-frame skipping of exon 22, predicted to delete most of the zinc binding region of the helicase domain. We suggest that including the WRN gene in genetic analysis of early onset diabetes, lipodystrophy or dyslipidaemia would allow for the opportunity to diagnose some cases of Werner syndrome long before clinical criteria are met, thereby allowing early implementation of important primary prevention interventions. Werner syndrome is a premature ageing disorder caused by biallelic variants in the WRN gene. WRN encodes a dual DNA helicase/exonuclease enzyme. Molecular diagnosis is commonly only made at a late disease stage in the third or fourth decade, when cardinal features have become apparent. We describe a 28 year-old woman who presented with early onset diabetes associated with partial lipodystrophy, severe dyslipidaemia and rapidly progressive liver fibrosis related to non-alcoholic steatohepatitis in the absence of progeroid features. Werner syndrome was diagnosed by trio exome analysis, which revealed compound heterozygous WRN mutations: the known variant c.1290_1293del (p.Asn430Lysfs*7) and the novel intronic splice site variant c.2732+5G>A. cDNA analysis demonstrated this to lead to in-frame skipping of exon 22, predicted to delete most of the zinc binding region of the helicase domain. We suggest that including the WRN gene in genetic analysis of early onset diabetes, lipodystrophy or dyslipidaemia would allow for the opportunity to diagnose some cases of Werner syndrome long before clinical criteria are met, thereby allowing early implementation of important primary prevention interventions. IntroductionLipodystrophy denotes loss of adipose tissue from some or all adipose depots, and may be genetic or acquired. It is characteristically not reversible even given sustained positive energy balance, and it is usually accompanied by metabolic features of “adipose failure” such as insulin resistance, fatty liver, dyslipidaemia and often diabetes. These, in turn, strikingly increase risk of advanced liver disease and accelerate atherosclerosis.1Brown R.J. Araujo-Vilar D. Cheung P.T. et al.The Diagnosis and Management of Lipodystrophy Syndromes: a Multi-Society Practice Guideline.J Clin Endocrinol Metab. 2016; 101: 4500-4511Google Scholar In cases of partial lipodystrophy, clinical signs are most commonly first noticed peripubertally, especially in girls.Werner syndrome (WS; OMIM #277,700) is an autosomal recessive disorder characterized by short stature due to absent of the pubertal growth spurt and a wider pattern of premature ageing features with onset on the third decade. These include greying and thinning of the hair, bilateral cataracts, scleroderma-like skin changes, cutaneous calcinosis and ulceration of the extremities, premature atherosclerosis and a propensity to develop early onset cancers. WS patients may also develop partial lipodystrophy affecting subcutaneous fat, and show premature muscle loss from the limbs, or sarcopenia. Diabetes, fatty liver disease and dyslipidaemia are commonly reported amongst those with an established dagnosis.2Oshima J. Martin G. Hisama F. Werner Syndrome.GeneReviews. 2002; https://www.ncbi.nlm.nih.gov/books/NBK1514/?report=readerGoogle Scholar Diagnostic criteria have been developed,3Takemoto M. Mori S. Kuzuya M. et al.Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey.Geriatr Gerontol Int. 2013; 13: 475-481Google Scholar, 4Oshima J. International Registry of Werner Syndrome: diagnostic Criteria2000:http://www.pathology.washington.edu/research/werner/registry/diagnostic.html.Google Scholar but they rely on several of the later onset features of the disease, and so diagnosis is often made only at late stages of the natural history. Delays of many years between appearance of the first clinical manifestation and the molecular diagnosis are thus the norm.5Koshizaka M. Maezawa Y. Maeda Y. et al.Time gap between the onset and diagnosis in Werner syndrome: a nationwide survey and the 2020 registry in Japan.Aging (Albany NY). 2020; 12: 24940-24956Google ScholarIsolated partial lipodystrophy is not regarded as a classic sign of WS, and its diagnosis in childhood rarely triggers targeted analysis of the WRN gene.6Donadille B. D'Anella P. Auclair M. et al.Partial lipodystrophy with severe insulin resistance and adult progeria Werner syndrome.Orphanet J Rare Dis. 2013; 8: 106Google Scholar However genetic diagnosis increasingly commonly relies on sequencing of exomes or panels of relevant genes, and inclusion of the WRN gene in these panels presents a key opportunity to accelerate diagnosis and to open up opportunities for early interventions and trials seeking to delay or prevent later complications.Here, we report a woman first diagnosed with early onset diabetes at 10 years old, later developing anatomical and biochemical features of partial lipodystrophy together with fibrosing steatohepatitis with no overt features of premature ageing. Exome sequencing revealed that she is affected by WS, with compound heterozygous WRN mutations including a novel splice variant predicted to produce a WRN protein missing the zinc binding region of the helicase domain.Materials and methodsExome sequencing and analysisGenomic DNA was isolated from leucocytes using Qiaamp DNA mini kit (Thermoscientific™). Exome-enriched genomic DNA (gDNA) libraries were prepared with the Illumina TruSeq Exome Library (FC-150–1002) and sequenced on the NextSeq 550 platform (llumina Inc, #SY-415–1002) with the NextSeq 500/550 Mid-Output v2.5 150 cycle kit (20,024,904; 2 × 75 bp sequencing). Variant calling was performed to GATK best practice standards7Van der Auwera GaBOC. Genomics in the Cloud: Using Docker, GATK, and WDL in Terra.:O'Reilly Media.; 2020.Google Scholar with Base Quality Score Recalibration, realignment, and HaplotypeCaller variant calling, with bcbio.8Chapman B., Kirchner, R., Pantano, L., et al. 2020.Google Scholar Sequences were aligned with bwa9H. L. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM.2013.Google Scholar to the GRCh38/hg38 human reference genome10Schneider V.A. Graves-Lindsay T. Howe K. et al.Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.Genome Res. 2017; 27: 849-864Google Scholar and variant effects were annotated via Ensembl Variant Effect Predictor. Candidate causal variants were identified by applying custom filters to variant tables following GATK guidelines,7Van der Auwera GaBOC. Genomics in the Cloud: Using Docker, GATK, and WDL in Terra.:O'Reilly Media.; 2020.Google Scholar and were confirmed by Sanger sequencing of trio gDNA. PCR primers were designed with PrimerBlast and Primer3Plus. Amplicons were purified from agarose gel by QIAquick Gel Extraction kit and sequenced in both directions by SourceBioscience. Sequencing traces were assessed by SnapGeneViewer. Splicing effects of the intronic mutation identified mutation were predicted with the Alternative Splice Site Predictor (ASSP) Tool.11Wang M. Marin A. Characterization and prediction of alternative splice sites.Gene. 2006; 366: 219-227Google ScholarCell culture and cDNA sequencingPrimary dermal fibroblast cultures were established from a punch skin biopsy from the proband, according to standard procedures. Fibroblasts were maintained in DMEM (41,966–029; LifeTechnologies) supplemented with 10% foetal bovine serum (10,500,064; Gibco) and 1x Penicillin-Streptomycin-Glutamine (10,378,016; LifeTechnologies) at 37 °C in 5% CO2. For RNA analysis cells were trypsinised, pelleted by centrifugation and snap frozen on dry ice. Total cellular RNA was purified by Direct-zol RNA MiniPrep-Plus (R2072; Zymo). cDNA was prepared by High-Capacity cDNA kit (4,368,814; Applied Biosystems), and selected regions amplified by PCR with custom primers. Sanger sequencing of PCR products was undertaken by Source Bioscience.ResultsCase reportA 28-year-old female was the third child of non-consanguineous parents from Cape Verde. She was born at term (2800 gr) and her growth and developmental milestones were normal. Around age 7, she had acute weight loss. At that time, a postive HBeAg hepatitis B infection was diagnosed and treatment with pegylated interferon was attempted in Cape Verde. When she arrived in Switzerland at age 10, she had short stature with a moon face, a prominent abdomen and thin extremities. Laboratory workup found marked hyperglycaemia and a diagnosis of early onsed diabetes was made. Anti-glutamic acid decarboxylase (GAD) and anti-islet antigen 2 (IA2) antibodies were negative. High doses of insulin were needed to achieve glycaemic control. Recurrent episodes of compulsive hyperphagia were reported which contributed also to the hyperglycaemia. In spite of pubertal development with menarche at age 13, she did not show a growth spurt. In ensuing years she was noticed to have severe dyslipidaemia, with serum triglycerides up to 100 mmol/L and low HDL cholesterol of 0.3 mmol/L. She had been treated, since the age of 19 years, with pegylated alpha-interferon, entecavir, tenofovir and finally, tenofovir alafenamide due to HBeAg positive chronic hepatitis B. Otherwise, she had been treated with levothyroxine for autoimmune hypothyroidism since the age of 28 years.Physical examination at age 28 years (Fig. 1) revealed height to be 147 cm (−2.5 SD; target height 159.3 ± 8.5 cm), weight 32.25 kg (−3.8 SD) and body mass index (BMI) 14.9 kg/m2 (−3.96 SD).12Institute CsHoP-R. Pediatric Z-Score Calculator:https://zscore.research.chop.edu/resultbmi.php.Google Scholar The abdomen was prominent without palpable hepatomegaly or clinical signs of chronic liver disease. Limbs appeared thin due to loss of both subcutaneous adipose tissue and muscle, with no evidence of the calf hypertrophy seen in some commoner forms of familial partial lipodystrophy (FPLD) such as FPLD2 or FPLD3. The skin on the extremities appeared tight. The thin limbs contrasted with her round face that gave her a youthful appearance (Fig. 1). Mild facial hirsutism was observed. There was no greying or thinning of the hair, evidence of was revealed at and dyslipidaemia, with serum cholesterol triglycerides mmol/L and HDL cholesterol mmol/L was at F. M. et and with in and female adult of different body Clin Endocrinol Metab. Scholar were liver without hepatomegaly and identified of cm in biopsy was performed both in the liver and in the The liver features of with and and a of without hepatitis was also found in the some of the was both in with and in it a pattern of in the liver biopsy performed 10 years some features were but without fibrosis (Fig. The to an and was no sign of and were found by in some and The steatohepatitis and fibrosis not be by hepatitis biopsy at the age of years and no or fibrosis and biopsy at years revealed and and fibrosis of adipose tissue from the limbs to fatty liver, dyslipidaemia and insulin diabetes to a clinical diagnosis of partial genetic was given the in the adipose to the pattern of partial lipodystrophy, which usually in the face and to the M. R.J. of Clin 2017; Scholar Diagnostic sequencing of a of in disorder and onset of diabetes of the was undertaken no variant was found and the diagnostic was to exome sequencing and cDNA exome analysis of the and her parents to genetic for the lipodystrophy with no de novo or biallelic mutations in or splice S. L. et al.The of WRN mutations in Werner syndrome 2006; 27: Scholar heterozygous variant in the WRN gene from the was identified (Fig. mutations in the WRN gene WS, the mutation identified is predicted to the WRN protein before the helicase and the pattern of adipose and muscle loss in the was of mutation was thus in the sequence at the WRN a variant was a novel intronic splice site variant at the of was and to be from the (Fig. of the from and heterozygous WRN mutations in the splice site is the most sequence in P. Alternative Splicing that in 2017; 8: Scholar and the mutation the sequence to such the that it is no a human of the site is predicted to in loss of exon due to between the splice site in with the before exon Sequencing of cDNA from dermal from the revealed at the of the with by the (Fig. It also confirmed skipping of exon in the (Fig. It was not the protein is but it it is predicted to lead to in-frame of a of the zinc binding that the DNA helicase of WRN the of compound heterozygous mutations in the WRN gene confirmed the diagnosis of genetic the were assessed but not did not clinical or of analysis in the A. Sequencing of an in genomic DNA from the trio as and in DNA from the RNA as from the B. cDNA sequencing exon in and proband, skipping of exon in proband, by agarose gel PCR products and Sanger sequencing traces of WRN predicted in-frame from caused by exon binding DNA binding and Genomics clinical the diagnosis of WS, and was by of its clinical features and natural history. cutaneous calcinosis were was and did not or revealed treated by given sustained low serum to target The was and an of and of with the following The mass was on and of long insulin given to had an positive on insulin resistance but was due to weight which was regarded as given the low body mass index In spite of treatment with and triglycerides 100 at due to triglycerides from to to 100 and cholesterol from to to to with adipose the that human may be to the metabolic in this was by the in as a to the of in lipodystrophy the first treatment for this in It was in at by subcutaneous later she reported a of The insulin by from to serum triglycerides from to and cholesterol from to and clinical for of and liver are in is with of cases in it and is it a of early and on affected and also it several key features of ageing in that it may be a for of D. T. progeroid human of the WRN gene is a protein with the dual to DNA using a helicase and to from of DNA in which the is using an et and molecular an in DNA 2006; 13: Scholar It a in genome and of of the genome such as regions genes, or regions with long of or A. M. The Werner syndrome protein is from the syndrome protein by its to DNA Scholar These sequences are all to such as DNA or K. Y. Y. et for of a by an of human protein 2016; V.A. in DNA and Scholar The WRN protein these to prevent to DNA or between and is of the most in the progeroid features and of WS in S. WRN at for and M. of body in syndrome: for Scholar, J. Hisama and novel genetic to and Werner or variants in WRN have been to that lead to loss of the at the of the WRN protein J. R.J. Werner syndrome: clinical and 2017; Scholar so are have been S. L. et al.The of WRN mutations in Werner syndrome 2006; 27: Scholar In the we describe the WRN mutation was not at the the mutation leads to of in which exon is We have not established the WRN protein is but this protein would have a of of a which the helicase and a in with DNA WRN S. et of the helicase of Werner a key target in Scholar both the helicase and the are It is to that loss of only of the of the WRN protein may the of some cardinal features of WS in the we at to the age of of of the biochemical of the and up of the or with mutations be to to clinical in WS from late diagnosis in many diagnostic criteria have been in both the J. International Registry of Werner Syndrome: diagnostic Criteria2000:http://www.pathology.washington.edu/research/werner/registry/diagnostic.html.Google Scholar and M. Mori S. Kuzuya M. et al.Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey.Geriatr Gerontol Int. 2013; 13: 475-481Google Scholar diagnosis relies on cardinal features of WS that not usually the third or fourth decade. These include bilateral cataracts, or thinning and greying of a Japanese a age of onset of of WS of years, but a age at diagnosis of M. Maezawa Y. Maeda Y. et al.Time gap between the onset and diagnosis in Werner syndrome: a nationwide survey and the 2020 registry in Japan.Aging (Albany NY). 2020; 12: 24940-24956Google Scholar at the age of 28 years, the we describe did not criteria for WS and a youthful appearance of her face was on at The age of in WS been reported to be J. R.J. Werner syndrome: clinical and 2017; Scholar due often to early onset and A. R.J. and risk of in Werner syndrome: a 2013; 8: Scholar or of atherosclerosis such as It would be to with WS early in to and primary prevention of WS reported is the absence of a pubertal growth to adult this is not to according to the diagnostic the onset of the disease before the age of 10 is in a of diagnostic as J. International Registry of Werner Syndrome: diagnostic Criteria2000:http://www.pathology.washington.edu/research/werner/registry/diagnostic.html.Google Scholar the insulin resistance, diabetes, dyslipidaemia and fatty liver are as of M. Mori S. Kuzuya M. et al.Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey.Geriatr Gerontol Int. 2013; 13: 475-481Google Scholar In this diabetes was diagnosed at 10 years old, we have onset of severe insulin resistance as early as years in a reported of M. Mori S. Kuzuya M. et al.Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey.Geriatr Gerontol Int. 2013; 13: 475-481Google Scholar In both cases a genetic diagnosis was based on exome of metabolic seen in WS that of partial lipodystrophy, in with the progressive loss of subcutaneous adipose tissue from the anatomical evidence of lipodystrophy may not be at the of of metabolic when lipodystrophy it is usually associated with loss of muscle mass that criteria for M. Mori S. Kuzuya M. et al.Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey.Geriatr Gerontol Int. 2013; 13: 475-481Google Scholar is the hypertrophy commonly seen in P. B. A. muscle in with familial partial lipodystrophy, Metab. 2013; Scholar of adipose tissue may a as in of B. D'Anella P. Auclair M. et al.Partial lipodystrophy with severe insulin resistance and adult progeria Werner syndrome.Orphanet J Rare Dis. 2013; 8: 106Google diagnosis increasingly relies on sequencing of of related to a of using to such panels is and and we suggest that including WRN in panels to diabetes, or lipodystrophy would accelerate diagnosis of WS in some would of a including and at an early years of the of WS since of WRN no clinical trials in WS are as and of patients at a disease stage when may have a of may to to the disease is only of clinical of in patients with lipodystrophy, it is to that WS patients have “adipose K. A. A. a for the of adipose 101: Scholar subcutaneous adipose tissue a for hypertrophy and in a of the adipose tissue and an of at the increase and a at the liver, muscle and to metabolic and insulin The severe and rapidly fibrosis on steatohepatitis in together with the development of of its an the of liver of lipodystrophy, which may be in Werner low those patients should of insulin and that adipose and are all of treatment with some L. et in a for Werner J. Scholar, M. K. to Management for Werner syndrome Gerontol Int. Scholar, K. S. K. et and in Werner syndrome by Scholar been to the metabolic in patients with and partial A. R.J. of in Lipodystrophy to Clin Endocrinol Metab. A. A. S. Lipodystrophy 2020; 13: Scholar which was by metabolic is a premature ageing syndrome that and diagnosis implementation of and and clinical diagnostic criteria lead to late diagnosis in most for WRN mutations in insulin diabetes, dyslipidaemia lipodystrophy presents the opportunity for diagnosis based on these metabolic features long before appearance of the of is by the from in the or this and and clinical and performed genetic analysis and liver biopsy and the and the and the were undertaken on a clinical diagnostic in with and The and her parents for genetic was also for IntroductionLipodystrophy denotes loss of adipose tissue from some or all adipose depots, and may be genetic or acquired. It is characteristically not reversible even given sustained positive energy balance, and it is usually accompanied by metabolic features of “adipose failure” such as insulin resistance, fatty liver, dyslipidaemia and often diabetes. These, in turn, strikingly increase risk of advanced liver disease and accelerate atherosclerosis.1Brown R.J. Araujo-Vilar D. Cheung P.T. et al.The Diagnosis and Management of Lipodystrophy Syndromes: a Multi-Society Practice Guideline.J Clin Endocrinol Metab. 2016; 101: 4500-4511Google Scholar In cases of partial lipodystrophy, clinical signs are most commonly first noticed peripubertally, especially in girls.Werner syndrome (WS; OMIM #277,700) is an autosomal recessive disorder characterized by short stature due to absent of the pubertal growth spurt and a wider pattern of premature ageing features with onset on the third decade. These include greying and thinning of the hair, bilateral cataracts, scleroderma-like skin changes, cutaneous calcinosis and ulceration of the extremities, premature atherosclerosis and a propensity to develop early onset cancers. WS patients may also develop partial lipodystrophy affecting subcutaneous fat, and show premature muscle loss from the limbs, or sarcopenia. Diabetes, fatty liver disease and dyslipidaemia are commonly reported amongst those with an established dagnosis.2Oshima J. Martin G. Hisama F. Werner Syndrome.GeneReviews. 2002; https://www.ncbi.nlm.nih.gov/books/NBK1514/?report=readerGoogle Scholar Diagnostic criteria have been developed,3Takemoto M. Mori S. Kuzuya M. et al.Diagnostic criteria for Werner syndrome based on Japanese nationwide epidemiological survey.Geriatr Gerontol Int. 2013; 13: 475-481Google Scholar, 4Oshima J. International Registry of Werner Syndrome: diagnostic Criteria2000:http://www.pathology.washington.edu/research/werner/registry/diagnostic.html.Google Scholar but they rely on several of the later onset features of the disease, and so diagnosis is often made only at late stages of the natural history. Delays of many years between appearance of the first clinical manifestation and the molecular diagnosis are thus the norm.5Koshizaka M. Maezawa Y. Maeda Y. et al.Time gap between the onset and diagnosis in Werner syndrome: a nationwide survey and the 2020 registry in Japan.Aging (Albany NY). 2020; 12: 24940-24956Google ScholarIsolated partial lipodystrophy is not regarded as a classic sign of WS, and its diagnosis in childhood rarely triggers targeted analysis of the WRN gene.6Donadille B. D'Anella P. Auclair M. et al.Partial lipodystrophy with severe insulin resistance and adult progeria Werner syndrome.Orphanet J Rare Dis. 2013; 8: 106Google Scholar However genetic diagnosis increasingly commonly relies on sequencing of exomes or panels of relevant genes, and inclusion of the WRN gene in these panels presents a key opportunity to accelerate diagnosis and to open up opportunities for early interventions and trials seeking to delay or prevent later complications.Here, we report a woman first diagnosed with early onset diabetes at 10 years old, later developing anatomical and biochemical features of partial lipodystrophy together with fibrosing steatohepatitis with no overt features of premature ageing. Exome sequencing revealed that she is affected by WS, with compound heterozygous WRN mutations including a novel splice variant predicted to produce a WRN protein missing the zinc binding region of the helicase domain.

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