ORIGINAL CONTRIBUTION Using Exome Sequencing to Reveal Mutations in TREM2 Presenting as a Frontotemporal Dementia–like Syndrome Without Bone Involvement Rita Joa˜o Guerreiro, PhD; Ebba Lohmann, MD; Jose´ Miguel Bra´s, PhD; Jesse Raphael Gibbs, MS; Jonathan D. Rohrer, MRCP; Nicole Gurunlian, MS; Burcu Dursun, MD; Basar Bilgic, MD; Hasmet Hanagasi, MD; Hakan Gurvit, MD; Murat Emre, MD; Andrew Singleton, PhD; John Hardy, PhD Objective: To identify new genes and risk factors as- Results: In 3 probands with FTD-like disease, we iden- sociated with frontotemporal dementia (FTD). Several tified different homozygous mutations in TREM2 that had genes and loci have been associated with different forms previously been associated with polycystic lipomembra- of FTD, but a large number of families with dementia do nous osteodysplasia with sclerosing leukoencephalopa- not harbor mutations in these genes. thy (PLOSL). None of these 3 patients had a typical clini- cal presentation of PLOSL: they presented with behavioral Design: Whole-exome sequencing and whole-genome change and subsequent cognitive impairment and mo- genotyping were performed in all patients. Genetic vari- tor features but without any bone cysts or bone- ants obtained from whole-exome sequencing were inte- associated phenotypes. Imaging showed white matter ab- grated with the data obtained from whole-genome geno- normalities as well as frontal atrophy in all 3 patients. typing. Conclusions: Our results show that TREM2 is respon- sible for an unexpectedly high number of dementia cases Setting: Database of the Behavioral Neurology Outpa- in our cohort, suggesting that this gene should be taken tient Clinic of the Department of Neurology, Istanbul Fac- into account when mutations in other dementia genes ulty of Medicine, Istanbul, Turkey. are excluded. Even for complex syndromes such as de- mentia, exome sequencing has proven to be a rapid and Patients: Forty-four Turkish patients with an FTD- cost-effective tool to identify genetic mutations, allow- like clinical diagnosis were included in the study. Rela- ing for the association of clinical phenotypes with un- tives were screened when appropriate. expected molecular underpinnings. Author Aff of Neuroge Main Outcome Measure: Mutations in the trigger- JAMA Neurol. 2013;70(1):78-84. Published online October Institute on ing receptor expressed on myeloid cells 2 gene (TREM2). 8, 2012. doi:10.1001/jamaneurol.2013.579 Institutes o Maryland ( and Singlet Center for N ENETIC ANALYSIS HAS these variants, they will not be identifiable Cell Biolog identified several genes through genome-wide association studies. Coimbra, C and loci as underlying Technological advances now allow the (Drs Guerr frontotemporal demen- analyses of complete exomes and ge- Lilla Westo tia (FTD). These in- nomes, placing us in a position to study Departmen Neuroscien clude mutations in the granulin precur- these families. In fact, several recent stud- Neurology G 1,2 sor gene (GRN, OMIM 138945) and ies have succeeded in identifying the ge- and Hardy, microtubule-associated protein tau gene netic basis of different disorders by using Gurunlian) (MAPT, OMIM 157140).3 Most recently, exome and genome sequencing.6-9 These Neurodegen a hexanucleotide repeat expansion in the studies demonstrate that even by using a Dementia R noncoding region of the chromosome 9 small number of samples, it is now pos- UCL Institu University open reading frame 72 gene (C9ORF72, sible to uncover genetic alterations not only Queen Squ OMIM 614260) has been shown to be the in mendelian diseases but also in multifac- London, En genetic cause of disease in a group of pa- torial complex disorders. This has great Behavioral tients with FTD.4,5 Nonetheless, a large part clinical utility with major implications for Movement of the genetic cause of FTD remains un- disease gene discovery, diagnosis, and, ul- Departmen known. This is particularly evident in fami- timately, therapeutic approaches. Istanbul Fa (Drs Lohm lies with ages at onset between 55 and 70 As part of an ongoing study aimed at Hanagasi, G years where mendelian variants in yet to be characterizing, from a genetic perspec- and Institu Author Affiliations are listed at identified genes are thought to underlie the tive, a large sample of Turkish families with Medicine (D the end of this article. disease. Because of the low frequency of dementia, we sequenced the whole exome University, JAMA NEUROL/ VOL 70 (NO. 1), JAN 2013 WWW.JAMANEURO.COM 78 ©2013 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 of several cases. In 3 of these families, we identified ho- SANGER SEQUENCING mozygous mutations in the triggering receptor ex- pressed on myeloid cells 2 gene (TREM2, OMIM 605086). To confirm the mutations found by exome sequencing in the Mutations in this gene have previously been associated probands, test family members, and screen control individu- with polycystic lipomembranous osteodysplasia with scle- als, ExonPrimer (http://ihg.gsf.de/ihg/ExonPrimer.html) was rosing leukoencephalopathy (PLOSL) (also known as used to generate primers for amplification of TREM2 exon 2. This exon was polymerase chain reaction amplified using Roche Nasu-Hakola disease). This is a rare autosomal reces- FastStart PCR Master Mix polymerase (Roche Diagnostics Corp) sive disease characterized by early-onset progressive de- and the polymerase chain reaction products were sequenced mentia and bone cysts. Herein, we describe 3 Turkish pa- using the same forward and reverse primers with Applied Bio- tients with TREM2 homozygous mutations, presenting systems BigDye terminator version 3.1 sequencing chemistry clinically with an FTD-like syndrome but without any and run on an ABI3730XL genetic analyzer as per the manu- bone cysts or other bone phenotype, suggesting that mu- facturer’s instructions (Applied Biosystems). The sequences were tations in this gene may be a more frequent cause of de- analyzed using Sequencher software, version 4.2 (Gene Codes). mentia than previously considered and showing that this diagnosis should be considered even in the absence of ILLUMINA SNP BEADCHIP ANALYSIS bone problems. To exclude the presence of large structural variants and to per- form homozygosity mapping, the available DNA samples from METHODS the 3 families in whom mutations in TREM2 were found were run on HumanOmniExpress BeadChips as per the manufac- HUMAN SUBJECTS turer’s instructions (Illumina Inc). Data were visualized and ana- lyzed using the GenomeStudio Data Analysis Software (Illu- We reviewed the database of the Behavioral Neurology Out- mina Inc). patient Clinic of the Department of Neurology, Istanbul Fac- ulty of Medicine, Istanbul, Turkey to identify all patients with RESULTS a primary clinical syndrome within the FTD spectrum10 and an available blood sample. Forty-four patients were identified who had presented initially with either a change in personal- GENETIC ANALYSIS ity and behavioral symptoms (consistent with either probable or possible behavioral variant FTD following the Frontal Tem- No pathogenic mutations in the known FTD genes were poral Dementia Consortium criteria11) or with a progressive found in this cohort; however, in 1 sample, a novel vari- aphasia (consistent with either semantic dementia or progres- ant in MAPT was identified (data not shown). sive nonfluent aphasia), although a number of patients subse- By applying a series of filters, we were able to iden- quently developed atypical features including seizures. A di- tify a previously described homozygous nonsense mu- agnosis of FTD was made on the basis of clinical and neuropsychological assessment and independent of any neu- tation (p.Q33X) in TREM2 in case 1 (eTable 2). This led roimaging. Seventy-six samples from healthy controls (mainly us to screen the whole cohort for variants in TREM2.Two caregivers and spouses accompanying patients to the clinic) were more homozygous changes were identified. For these 3 also collected. Written consent for participation was obtained cases, whole-exome sequencing resulted in the identifi- in accordance with institutional review board standards. Most cation of an average of 1225 novel (not present in db- of the families had Turkish ethnic background (n=39) and sus- SNP version 132 or the 1000 Genomes project and in- pected (n=23) or known (n=4) consanguinity. The general fea- cluding variants located in exon/intron boundaries Ϯ4 tures of the patients’ cohort studied are presented in eTable 1 base pairs) single-nucleotide variants, of which an aver- (http://www.jamaneuro.com). Genomic DNA from these age of 70 were homozygous (eTable 2). SIFT was used samples was prepared from venous blood samples by standard as a first-pass filter to predict how the amino acid sub- procedures. Samples from the human genome resequencing panel of the Centre d’Etude du Polymorphisme Humain– stitutions found would affect protein function, taking into Human Genome Diversity Cell Line Panel (n=275) were used account sequence homology and the physical proper- to assess the frequency of genetic variation in exon 2 of TREM2.12 ties of amino acids, predicting 17 homozygous changes to be damaging in case 2 (including TREM2 p.T66M) and EXOME SEQUENCING 30 homozygous changes in case 3 (including TREM2 p.Y38C). From the analysis of the results correspond- Sequences corresponding to all annotated human exons were en- ing to stop-loss and stop-gain variants in the 3 probands riched by hybridization using the SeqCap EZ Exome Library ver- (Table), the stop-loss mutation p.X152R in PRB1, found sion 1.0 (Roche NimbleGen), as per manufacturers’ protocols. The in probands 1 and 3, could be considered as a possible DNA samples were sequenced in 1 flow cell lane each, on paired- cause of disease. However, this variant is now known to end 50–base pair HiSeq 2000 runs (Illumina Inc), yielding an av- be a polymorphism (rs111877208) with a high minor al- erage of about 6 billion high-quality bases per sample. Image analy- lele frequency in the 1000 Genomes project (minor al- sis and base calling were performed using the Illumina pipeline lele frequency A=0.273).