exome sequencing identified a splice site mutation in fhl1...

DOI: 10.1161/CIRCGENETICS.115.001193

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Exome Sequencing Identified a Splice Site Mutation in FHL1 that Causes

Uruguay Syndrome, an X-Linked Disorder with Skeletal Muscle Hypertrophy

and Premature Cardiac Death

Running title: Xue et al.; Mutation in FHL1 causes Uruguay syndrome

Yuan Xue, PhD1; Benedikt Schoser, PhD2; Aliz R. Rao, PhD3; Roberto Quadrelli, MD4; Alicia

Vaglio, MD4; Verena Rupp, BS5; Christine Beichler, BS5; Stanley F. Nelson, MD3; Gudrun

Schapacher-Tilp, PhD6; Christian Windpassinger, PhD5; William R. Wilcox, MD, PhD7

1Emory Genetics Laboratory, 7Division of Medical Genetics, Department of Human Genetics, Emory University, Atlanta, GA; 2Friedrich-Baur Institut, Neurologische Klinik, Klinikum der

Universität, München, Germany; 3Department of Human Genetics, UCLA School of Medicine, Los Angeles, CA; 4Instituto de Genetica Medica, Hospital Italiano, Montevideo, Uruguay;

5Institute of Human Genetics, Medical University of Graz; 6Department for Mathematics and Scientific Computing, Karl-Franzens-University Graz, Graz, Austria

Correspondence:

Yuan Xue, PhD

Emory University

1533 Richard Stokes Dr.

Decatur, GA 30033

Tel: (310) 739-4985

E-mail: [email protected]

Journal Subject Terms: Basic Science Research; Mechanisms; Genetics; Hypertrophy; Cardiomyopathy

Vaglio, MD ; Verena Rupp, BS ; Christine Beichler, BS ; Stanley F. Nelson, MDMDMD ; GuGuG drdrunun

Schapacher-Tilp, PhD6; Christian Windpassinger, PhD5; William R. Wilcox, MDMDMD, PhPhPhDDD7 7 7

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Abstract:

Background - Previously we reported a rare X-linked disorder, Uruguay syndrome in a single

family. The main features are pugilistic facies, skeletal deformities, muscular hypertrophy

despite a lack of exercise and cardiac ventricular hypertrophy leading to premature death.

Methods and Results - An approximately 19Mb critical region on X chromosome was identified

through identity by descent analysis of three affected males. Exome sequencing was conducted

on one affected male to identify the disease-causing gene and variant. A splice site variant

(c.502-2A>G) in the FHL1 gene was highly suspicious among other candidate genes and variants.

FHL1A is the predominant isoform of FHL1 in cardiac and skeletal muscle. Sequencing cDNA

showed the splice site variant led to skipping of exons 6 of the FHL1A isoform, equivalent to the

FHL1C isoform. Targeted analysis showed that this splice site variant co-segregated with disease

in the family. Western-blot and immunohistochemical analysis of muscle from the proband

showed a significant decrease in protein expression of FHL1A. RT-PCR analysis of different

isoforms of FHL1 demonstrated that the FHL1C is markedly increased.

Conclusions - Mutations in the FHL1 gene have been reported in disorders with skeletal and

cardiac myopathy but none has the skeletal or facial phenotype seen in patients with Uruguay

syndrome. Our data suggest that a novel FHL1 splice site variant results in the absence of

FHL1A and the abundance of FHL1C, which may contribute to the complex and severe

phenotype. Mutation screening of the FHL1 gene should be considered for patients with

uncharacterized myopathies and cardiomyopathies.

Key words: candidate genes; cardiac hypertrophy; gene; gene mutation; single nucleotide polymorphism; exome sequencing; gene discovery; Uruguay syndrome; skeletal deformities; cardiac ventricular hypertrophy

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Introduction

We previously described the Uruguay facio-cardio-musculo-skeletal syndrome, a rare X-linked

disorder found in a single family1 (OMIM#300280). No other families with this disorder have

been reported. The main features in affected males are skeletal muscular hypertrophy despite of

lack of exercise and cardiac ventricular hypertrophy leading to premature death. Other findings

include a distinctive facial appearance and skeletal abnormalities consisting of large, deformed

hands and feet, congenital hip dislocations, and scoliosis. The mother of the proband has a milder

phenotype while other women in the family are asymptomatic.

In this study, we performed an identity-by-descent genetic analysis and exome

sequencing to identify the causal gene of Uruguay syndrome, FHL1, broadening the significant

phenotypic variability found with mutations in FHL1. Further expression studies on muscle and

cultured myocytes demonstrate how the identified mutation may be causing some aspects of the

phenotype.

Materials and Methods

Patients

Informed consent was obtained from the patients for participation in research. This study was

approved by the human subjects research committees at Cedars-Sinai Medical Center (where YX

and WRW were through 2013) and the Facultad de Medicina, Universidad de la República

Oriental del Uruguay. Blood and muscle (trapezius) samples were taken from the proband VI-2.

Other family members only provided a blood sample from which DNA was isolated.

An abbreviated, updated pedigree as of 2014 is presented in Figure 1, retaining the

numbering in the original pedigree.1 The proband VI-2 graduated from college and is teaching

school. His hypertrophic cardiomyopathy remains stable on a beta blocker and he has developed

equencing to identify the causal gene of Uruguay syndrome, FHL1, broadening tthehehe ssigigignininififificacac nt

phenottypypypic vvara iaabibility found with mutations in FHHL1L . Further expressioon n studies on muscle and

cucuultttuured myooocycycytetetesss deeemomomonsnsnstrtrt atteee hhhowowow tthehehe iiideeentntntififi ied mmmutaaatititiononon mamm y y y bebebe cccauauusisisingnn sooomememe aaaspspspececectststs ooofff thththe e

phphhenennotype.

Materials and Methththodododsss



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no new orthopedic problems than those originally defined. VI-1 remains healthy and has had a

normal daughter. VI-3 also remains healthy and has a normal son. The proband’s affected

uncle, V-7, died at 48 years of age from heart failure. The mother of the proband who had minor

features of the disorder, V-2 is being evaluated for a myopathic syndrome associated with a

polyneuropathy.

Identification of the gene and the mutation

Genome-wide SNP analysis

A genome-wide 250K NspI Affymetrix SNP microarray genotype analysis (Affymetrix, CA,

USA) analysis was performed on three affected males (see Figure 1, pedigree members V-7, V-

12 and VI-2) at the Microarray facility at UCLA (University of California, Los Angeles). SNP

microarray gene chip data were subsequently analyzed with dCHIP software

(http://www.hsph.harvard.edu/cli/complab/dchip/).

Exome sequencing and bioinformatics analysis

Whole-exome sequencing (WES) was performed at BGI (Beijing Genomics Institute; Shenzhen,

China) on one affected male (patient VI-2; see Figure 1). Briefly, the Agilent SureSelect Human

All Exon Kit was used to enrich for 50 megabases of coding sequences. A capture kit restricted

to the X chromosome was not available at the time when the experiment was being performed.

The captured and amplified library was loaded onto an Illumina HiSeq 2000 platform for

sequencing at an estimated 50X coverage. Raw reads were aligned to the human reference

genome GRCh37.69 using SOAP (Short Oligonucleotide Analysis Package) which was also used

for variant calling. We identified regions shared by all three affected patients based on the

genotyping array analysis and selected regions on the X chromosome that did not have a

breakpoint for at least 1.5 million base pairs for further investigation in the exomic data. Variants

12 and VI-2) at the Microarray facility at UCLA (University of California, Los AAAngnggelelleseses).).). SSSNPNPNP

microaarrr ayayy gggene e chchip data were subsequently anallyzyzy ed with dCHIP softwaw re

hhhttppp://www.hshhsphphph.h.h. ararrvavavardrdrd ee.edudu/c/c/clilili/c/c/comomomplplplabaa /d/d/dchchchip/))).

ExExExomomome sequenccinnng andndnd bioinininfofof rmatata icccs anaaalyyysis

Whole-exome sequenenencicic ngg ((WEWEWES)S)S was pperformedd aaat t BGI (B(B(Beiee jij ngg Genennomomomics Institute; Shenzhen,

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were annotated using the software tool Variant Annotator X,2 which queries databases such as

the Ensembl Variant Effect Predictor.3 Public population databases [dbSNP135

(http://www.ncbi.nlm.nih.gov/SNP/), 1000 Genomes Project,4 EVS database

(http://evs.gs.washington.edu/EVS)] and in-house databases were used to filter sequencing

variants and identify novel single-nucleotide variants (SNVs) that are predicted to alter protein

function

Sanger sequencing

Sanger sequencing was conducted for validation of the variants found in candidate genes

identified through WES within the critical region in patient VI-2. After identifying the candidate

gene, FHL1, which contained a novel splice site variant, DNA from 15 available family

members were analyzed by Sanger sequencing for family segregation analysis. Sanger

sequencing was also performed on cDNA from patient VI-2 to analyze the end products of splice

site change. Primers were designed with the Primer3 program (primer sequences available on

request). PCR products were sequenced with the BigDye Terminator 3.1 Cycle Sequencing Kit

(Applied Biosystems, Foster City, CA,USA). Sequencing reactions were size-separated on the

ABI Prism 3100 DNA Analyzer (Applied Biosystems, Foster City, CA,USA), and sequence data

was collected with the ABI Data Collection software version 1.1 and subsequently analyzed with

the ABI DNA Sequencing Analysis version 3.6 software. The transcript of FHL1 NM_001449.4

was used for sequence alignment.

Functional study of the gene and variant

Western blot analysis

For western-blot studies, proteins were extracted from 50 mg of skeletal muscle from five

normal controls, and two disease controls (Becker muscular dystrophy, Duchenne muscular

gene, FHL1, which contained a novel splice site variant, DNA from 15 available e fafafamimimilylyy

memberers werer ananalyzed by Sanger sequencing forr ffama ily segregation annala ysis. Sanger

eeeququuencing wwwasss aaalslsl o o pepeperfrfrfororormemem d d d ononon cccDNDNDNA AA frfrfromomom paattieeent VVVI-I-I-2 2 2 tott aaannnalylylyzezez thththe ee ennd d d prprprodododucuu tststs ooof f f spspsplililicec

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equest). PCR producucuctststs were seseseququq ene ced d with the BBBiggDyye TeTeTermrmr inator 333.1.1.1 Cycy le Sequencing Kit

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dystrophy). Protein concentrations of the supernatants were measured with the BCA Protein

Assay Kit (Pierce). Samples were stored frozen at -80°C. Aliquots of protein (2 mg) were loaded

onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel, composed of 4% stacking (0.5 M Tris-

HCL [pH 6.8] and 10% SDS) and 10% resolving (1.5 M Tris-HCL [pH 8.8] and 12% SDS) gels.

The protein was then transferred to nitrocellulose membranes for 1hr (110V). Nonspecific

binding was blocked for 2 hr with TBST (10 mM Tris-HCL [pH 7.4], 140 mM NaCl, and 0.1%

Tween-20) containing 5% skimmed milk; this was followed by incubation with primary

antibodies for 4 hr at room temperature. Rabbit polyclonal antibody anti-FHL1 (AVIVA Systems

Biology) was used at a dilution of 1:5000 in TBST-5% skim milk. After washing, the

membranes were incubated with the corresponding anti-rabbit or anti-mouse IgG secondary

antibodies (Dako) at a dilution of 1:1000 for 1 hr at room temperature. The membranes were

developed with the chemiluminescence ECL system (Pharmacia) and then exposed to

autoradiographic film. Analysis of immunoreactive bands was performed semiquantitatively.

The used anti-rabbit polyclonal FHL1 antibody (dilution 1:2000 AVIVA Systems Biology)

recognizes the C-terminal domain of the protein and detects only FHL1A. As control maker we

used caveolin-3 at 21 kDa. The antibody is only able to bind isoform FHL1A. Even though

FHL1 different isoforms have common last coding exon but the C terminus actually are quite

different which is demonstrated nicely in figure 1C of review paper by Cowling (2011).

Immunohistochemistry

In brief, after fixation with 2% paraformaldehyde and blocking with 4% BSA in PBS, samples

were treated with a polyclonal anti-rabbit FHL1 antiserum (dilution 1:1000 AVIVA Systems

Biology) and monoclonal anti-mouse skeletal slow myosin antiserum (dilution 1:5000, Sigma,

Germany) overnight at 4°C. After rinsing for 15 min in PBS, the sections were incubated with

membranes were incubated with the corresponding anti-rabbit or anti-mouse IgGG G seseecooondndndararary y y

antibodidiese ((DaD koo))) at a dilution of 1:1000 for 1 hr aat t room temperature. ThThe membranes were

dededeveveveloped wiwiiththth ttthehehe cchehehemimimilululumiminenenescscscenene cecece EEECLCLCL sssysy teeemmm (PPhahaharmrmrmacaa iaaa) ) ) ananand dd ththhenenen expxpposososededed tttoo

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Cy3-conjugated streptavidin goat anti-rabbit serum (Jackson-Immuno Research, dilution 1:1000)

and with Alexa-Fluor 488 goat anti-mouse serum (Invitrogen). After the sections were mounted

onto slides, a Leica epifluorescent microscope with a Zeiss Axiovert imaging system was used

for visualization. For comparison with normal controls, as well as limb-girdle muscular-

dystrophy muscle, sections were prepared using identical procedures as described above.

RNA Isolation and Real-time PCR

Patient myoblasts were cultured in Skeletal Muscle Cell Growth Medium Supplement

(PromoCell) with 10% FBS (GIBCO), 50 μg/ml Gentamicin (GIBCO) and 7.5 ml Glutamax (per

500 ml of media) (GIBCO) at 37°C with 5% CO2. Cells were split 1:4 after reaching ~70%

confluency to avoid the generation of myotubes.

Cells were trypsinized with 0.05% Trypsin-EDTA (GIBCO) and cell pellets were washed

2x with 1x DPBS without calcium and magnesium (Life Technologies, GIBCO). RNA was

extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and eluted

in 14 μl RNase free H2O. 800 ng RNA of each sample were transcribed using Long-Range 2Step

RT-PCR Kit (Qiagen). cDNA concentrations were determined by NanoDrop measurement and

50 ng of each cDNA were then added to Fast Sybr Green Master Mix (Applied Biosystems, Life

Technologies) for semiquantitative real-time PCR. All qRT-PCR experiments were performed

on the StepOne PLUS Real time PCR System from (Applied Biosystems, Life Technologies).

Data were analyzed by using the Step One Plus Real time PCR software (Applied Biosystems)

and data were normalized to GAPDH. Numbering of the exons corresponds with

NM_001159702 (FHL1B, shown in Fig.1A) beginning with exon 3 as first coding exon. Primers

for FHL1 isoform expression analysis are listed in supplementary table 1. In general,

quantification experiments were performed in triplicates for each sample, except for the

confluency to avoid the generation of myotubes.

CCells wwerre e trypsinized with 0.05% Trypsinn-EDE TA (GIBCO) andd ccell pellets were washed

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expression of FHL1 A/B/C of the sample with the c.688+1G>A mutation, where due to a

technical error one dataset was excluded.

We performed exact Kruskal-

between the three groups (healthy control, Uruguay patient, and patient with a c.688+1G>A

mutation). In case of significant results we performed post-hoc pairwise comparison between the

healthy control and the patients with mutation using one-sided exact Mann-Whitney U tests.

Results

Hemizygosity mapping with dChip software detected an approximate 19 Mb shared disease

haplotype for all three affected males on Xq24-q26.3, which encompasses 708 consecutive SNPs

(rs1716767 [117,442,440; Xq24] to rs4829618 [136,780,188; Xq26.3]). Genomic coordinates are

based on the hg19/GRCh37 genome assembly.

Based on exome sequencing data, candidate genes located within the critical region had

single nucleotide change or insertion/deletion (see supplementary table 2). All the single base

pair nucleotide changes detected through exome sequencing were confirmed by Sanger

sequencing. Neither the insertion nor deletion was confirmed by Sanger, which might be due to

the limitation of next-generation sequencing technology on detecting insertions and deletions at

the time. All the single nucleotide changes except for a splice site variant in the FHL1 gene

(c.502-2A>G) were found with high allele frequency in the population databases, indicating they

are most likely benign polymorphisms. Sequencing of all available family members proved the

segregation of the FHL1 gene (c.502-2A>G) splice variant in the family according to a X-

chromosomal recessive inheritance pattern (see Figure 1).

Sequencing of cDNA derived from myoblasts of the Uruguay patient VI-2 proved

skipping of the entire exon 6. The resulting primary structure is identical to the protein isoform

haplotype for all three affected males on Xq24-q26.3, which encompasses 708 cooonsnsnsecececutututiviviveee SNSNSNPs

rs1716767 [117,442,440; Xq24] to rs4829618 [136,780,188; Xq26.3]). Genomic coordinates are

baaaseseeddd on theee hhhg19/GRCh37 genome assemblyy.

Based onnn eeexomememe sequeueuencn ing daatatata, candndndidattte genenees s s lololoccateddd wwwithinnn ttthee ccrrriticall l reeegiiionnn haaad

inglelel nnnucucuclleleotiddde e hchchanangege oro insesertrtrtiiion/deleleletititionon (((seeee ssupupplpllemeemennnttatary tttababablle 222))). AlAA l thththeee sisinglelel bbbasasee

pair nucleotide channngegegesss dededetetetectctctededed ttthrhrhrouououghghgh eeexoxoxomememe ssseqeqequeueuennncininnggg wewewererere ccconononfififirmrmrmededed bbyby Sanger



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FHL1C (figure 2B).

In Western blot experiments we showed that compared to a healthy control there is

almost complete absence of the FHL1A-protein (expected at 29 kDa) in our patients’ muscle

(Figure 3A). See supplemental figure for the other 4 controls and the Duchenne muscular

dystrophy and Becker muscular dystrophy controls. This is consistent with the observation that

we do not see any signal in the IF of tissue sections in muscle from the proband using an

antibody that detects the C-terminus of FHL1A (Figure 3B).

We tested the expression of three FHL1 isoforms (FHL1A, FHL1B and FHL1C) in

myoblasts from healthy individuals (control), patients with a c.688+1G>A mutation (the donor

splice site mutation seen in XMPMA patients) and the Uruguay patient (proband VI-2). By using

primers that span the exon junction of exon 5/6 we were also able to prove that exon 6 is missing

in our patients’ myoblasts and thus the expression of isoform A and B is markedly decreased.

The expression of isoform FHL1C, in which exon 6 and 7 are missing, is highly increased in

XMPMA and Uruguay patients. Interestingly, compared to the expression in the XMPMA

patient the level of FHL1C in the Uruguay patient is almost 4 fold increased. Exact Kruskal-

Wallis tests were significant for all isoforms (i.e. p=0.01, p=0.0035, and p=0.05 for FHL1A/B/C

(exon 3-4), FHL1C (exon 5-8), and FHL1A/B (exon 5-6), respectively). All post-hoc one-sided

pairwise comparisons between the healthy control and the Uruguay patient were significant.

Post-hoc one-sided pairwise comparisons between the healthy control and the patient with

c.688+1 mutation were significant for FHL1C (exon 5-8) and FHL1A/B (exon 5-6) (Figure 4).

Discussion

In this study, we identified FHL1 as the causative gene for the rare X-linked Mendelian disorder,

Uruguay syndrome, characterized in a previous publication,1 utilizing identity by descent

plice site mutation seen in XMPMA patients) and the Uruguay patient (proband d d VIVIVI-2-2-2).).). BBBy y y uusu iiing

primerrs s thhat sspapan n tht e exon junction of exon 5/6 wee wwere also able to prooveve that exon 6 is missing

nnn ooouur patientttss’s’ mmmyoyy blblblasasastststs aaandnd ttthuhuhus s s ththt eee exexexprprresesessiss on off isofofoforororm m m A aanand d d BBB isisis mmmarkekeedldldly y y deded crcrreaeaeasesesedd.d.

ThThThe ee expression ooof f isoffforrrm FHHHLLL1C, iiin nn whwhwhich h exxon 6 andndnd 777 aarerere missssiing, iss hhhiggghlhlly y incrrreeaeaseeedd d in

XMPMA and Uruguauauay y y papatiennntststs.. Interests inglg y,y,y commmpapared totoo tttheheh exppreessssioioion n in the XMPMA

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mapping, exome sequencing, and further genetic and expression studies. The c.502-2A>G splice

site mutation in FHL1 identified in Uruguay syndrome patients causes the skipping of exon 6

resulting in an isoform identical to FHL1C (Figure 2B). The abundance of isoform FHL1C in the

absence of FHL1A and B may explain the skeletal and cardiac muscle anomalies seen in

Uruguay patients.

Four and a half LIM domain protein 1 (FHL1) encodes a transcription factor protein with

highly conserved four-and-a-half tandem repeated LIM domains. The LIM domain is a protein-

interaction motif and is involved in linking proteins with both the actin cytoskeleton and

transcriptional machinery. Proteins containing LIM domain play important roles in various

cellular processes, such as cytoskeleton organization, signal transduction, gene expression and

cell differentiation.5 FHL1 (MIM#300163), located on Xq26.3, consists of 4-6 coding exons

(dependent on the isoform) and is expressed in skeletal and cardiac muscle cells,6 where it is

suggested to play a role in sarcomere synthesis and assembly. FHL1 has been identified as the

causative gene for six different X-linked myopathies, with patients often presenting with

cardiovascular disease.7 Different mutations in the FHL1 gene lead to different myopathies.

Since 2008, there are about 40 mutations reported in FHL1 related myopathies with a wide

spectrum of clinical phenotypes. It appears that different phenotype and severity may be due to

which LIM domain that FHL1 the mutation occurs and if all isoforms are affected (See review

by Schessl et al8 for more detail). Although these myopathies share some overlapping clinical

features, they differ with respect to age of onset, distribution and severity of the disease (see

review by Cowling et al9 and, Schessl et al8). It is unclear how different FHL1 mutations lead to

distinct muscle diseases.

There are at least three isoforms of FHL1 (FHL1A, 1B, 1C; see Figure 2A) generated by

cellular processes, such as cytoskeleton organization, signal transduction, gene expxpxprerr ssssssioioion n n anananddd

cell difffef rer nttiai tioon.n 5 FHL1 (MIM#300163), locateded on Xq26.3, consists oof 4-6 coding exons

dddepppendent oonnn thththeee isofofoforororm)m)m) aandndd isisis eeexpxpxprereressss ededd iiin nn skeeeleeetal aaandndnd cccara dididiacacac mmmusussclclcle ee ceellllllsss,6 whwhwherrreee ititit iiisss

uuugggggested to ppplayyy aaa rollle in saaarcrccomo ere ee syyynnnthesssisss anddd aaassesesembmbm lly.. FHHHLL1L1 hass bbbeennn iididentiiififieeded asss theee

causative gene for ssixixix dddifferentntnt XXX-linkeed myyoppathihihiese , withhh pppataa ients ofteteten n n presenting with

cacardrdioiovavascsculularar ddisiseaeasese..777 DiDiffffererenent t mumutatatitionons s inin tthehe FHFHL1L1 gggenenee leleadad tto o didiffffererenent t mymyyopoppatathihieses..



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11

alternative splicing. These isoforms differ in primary structure, expression pattern, binding

partners and subcellular locations. The FHL1A (commonly referred as FHL1) isoform contains

all exons except exon 7. The C-terminal sequence of full-length isoform FHL1B binds RBP-Jk.

Isoform FHL1C, with two and a half LIM domains, skips exons 6 and 7 with a similar C-

terminus as FHL1B. FHL1A is the predominant isoform in heart and skeletal muscle; FHL1B

and C are also expressed at a much lower level.6,10 Each of the FHL1 protein isoforms appears to

exhibit distinct functions. However, in striated muscle much less is known of the functions of

FHL1B and FHL1C. Both proteins are hypothesized to play an important role during myogenesis

via regulation of RBP-Jj function.9

In previous studies, two splice site mutations in FHL1 have been reported: one on the

splice acceptor site of exon 6, having the same location as the splice site mutation in our patient

(c.502-2A>T; Pen et al., 2015) and the other one in the splice donor site of exon 6

(c.688+1G>A)11. In both studies and the current study, the end product of the splice site mutation

is identical to isoform FHL1C. As expected, the expression of FHL1A and B are decreased and

FHL1C is significantly increased in all studies.12,13

The phenotypes of patients in these three reports are similar in terms of cardiac

hypertrophy, but otherwise are quite different (see Table 1 for comparison). X-linked myopathy

with postural muscle atrophy (XMPMA, MIM#: 300696) is a late-onset scapuloaxioperoneal

myopathy disorder characterized by postural muscle atrophy with rigid spine syndrome, pseudo-

athleticism due to muscular hypertrophy and hypertrophic cardiomyopathy.14 Two key features

of XMPMA, namely rigid spine and postural muscle atrophy, are not seen in Uruguay patients.

Uruguay syndrome has an earlier onset, skeletal deformities, and a pugilistic facial appearance,

not seen in XMPMA patients. Interestingly, the expression data showed that there is significant

In previous studies, two splice site mutations in FHL1 have been reportedd:d: oooneee ooon n n thththeee

plice aaccccepptotor siitete of exon 6, having the same loccatation as the splice site e mum tation in our patient

ccc.5550002-2A>TTT;;; PePePen n n ett aaall.l., , , 202020151 ) ) ) ananand d d thththe e e otototheeer r r onono e innn tthe spspsplililicecece dooonononor r r siss tetee ooof exxononon 666

ccc.6.668888 +1G>A))11. IIIn booothhh studddieieies and d d thhhe currrrennnt stttudddy,,, ttthehehe eeennd prrroddduct ooof ff thheee sspsplicee ssiitite mmmutaattiooon

s identical to isoforrm m m FHF L1C.C.C. AsAsA expece ted, the exexexprp essiononon ooof FHL1A A A ananand B are decreased and

FHFHL1L1C C isis ssigiggninifificacantntlylyy iincncrereasaseded iin n alalll ststududieies.s.121212,131313



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12

more FHL1C in Uruguay patients than in XMPMA patients. The higher level of FHL1C in

Uruguay patients may explain their more severe phenotype. The phenotype associated with the

altered splicing due to the c.502-2A>T mutation (Uruguay patient has c.502-2A>G) known as

Emery-Dreifuss muscular dystrophy type 6 (EDMD6) Plus phenotype is characterized by a

combination of muscular atrophy, pulmonary artery hypoplasia and facial dysmorphology.12 The

adult in their family (patient 3) has a similar facial appearance as patients affected with the

Uruguay syndrome. However, EDMD Plus patients develop weakness and limitations in the

movement of the neck not seen in the Uruguay syndrome. While EDMD Plus is associated with

brachydactyly and wide feet, the skeletal malformations are not as severe as those seen in the

Uruguay syndrome. A missense mutation in the third LIM domain of FHL1 has also been

reported in a family with contracture, rigid spine, and cardiomyopathy.15

Both gain of toxic function and loss of normal protein function mechanisms have been

postulated for the pathogenesis of FHL1 associated disorders.9 The toxic accumulation of protein

aggregates (i.e. Toxic gain of function) is suggested to be responsible for the diagnostic feature

of severe FHL1 associated reducing body-myopathy. Reducing body aggregates are observed in

skeletal muscle from a few patients with XMPMA,8,11 however not in EDMD Plus, or Uruguay

syndrome at least by light microscopy. The majority of FHL1 mutations in EDMD patients are

frameshifts, which are predicted to result in premature stop codons. These novel mRNA

transcripts serve either as templates for the expression of truncated protein products or loss of

expression due to nonsense mediated-decay.16 Loss of normal FHL1A protein is present in all the

disorders. XMPMA and Uruguay syndrome have muscular hypertrophy and elevated FHL1C

protein levels. Although FHL1C expression is also increased in EDMD, muscular hypertrophy

has not been reported. Why the phenotypes of the 3 disorders are significantly different in many

Uruguay syndrome. A missense mutation in the third LIM domain of FHL1 has aalalsosoo bbbeeeeeen n n

eporteded iin a faf mimilyl with contracture, rigid spine, anand cardiomyopathy.15

Both gagagaininin ooof tototoxixix ccc fufufunccctititiononon aaandndd lllosoo s ss ofofof normamm l prprprotototeieiein nn fuuunnnctititionono mememechanannisisismsmsms hhavavveee bebebeenenen

popoostttuluu ated for thhee ppathhhogggenesssisss of FHHHLL1 assococociateddd disososordrdrderrrs...9 Thehehe toxiccc aaaccccumumumulatioioionnn oofof pprottteiin

aggregates (i.e. Toxxicicic gagagain of f f fufufuncnction) ) is suggggeg stttedede to be rrresesespoponsiblee fffororor the diagnostic feature

ofof sseveverere e FHFHL1L1 asassosociciatateded rrededucucining g g bobodydyy-mmyoyoy papap ththy.y.y RRededucucining g g bobodydyy aaggggggreregagag tetess arare e obobseservrveded iin ndd



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13

respects cannot be explained on the basis of gain of function due to overexpression of FHL1C,

unless there is a significant difference in the amount of FHL1C protein produced among the

disorders. It is interesting to note that there is a missense variant in the BCORL1 gene identified

in Uruguay patient (see suppl. Table 2). BCORL1 can act as a corepressor when tethered to DNA.

To date, this gene has not been associated with any disease. Variants in this gene have only been

found in individuals with autism and intellectual disability. Even though the variant in this gene

is predicted to be deleterious by computational algorithms and this gene is expressed in heart and

muscle, we are not sure how the variant in this gene can contribute to the phenotype seen in

Uruguay patients or any patients with muscular disorders.

In conclusion, we have identified the FHL1 gene as the causative gene for Uruguay

syndrome. Our study demonstrated that the splice site mutation of FHL1 detected in patients

with Uruguay syndrome leads to abundance of isoform FHL1C and absence of other isoforms,

especially FHL1A, in skeletal muscle what might explain skeletal muscle hypertrophy and

cardiac dysfunction. The findings of the current study have important implications for diagnostic

evaluation, screening, and genetic counseling of patients (also carriers) with myopathy of an

unknown genetic cause, particularly in cases where pedigree information would suggest X-linked

inheritance.

Acknowledgments: We wish to express our gratitude to the patients and their family for

participating in this study. We thank Denise Salazar and Kathy Porpora for their technical

support.

Conflict of Interest Disclosures: None.

In conclusion, we have identified the FHL1 gene as the causative gene fororr UUUruuuguguguayayay

yndroomeme. OuOur sttudu y demonstrated that the splice ssitite mutation of FHL11 ded tected in patients

wwwithhh Uruguayyy sssynynyndrdd omomomeee leleleadada s tototo aaabububundndndanananceee ooof ff isofooorm m FHFHFHL1L1L1C ananand d d ababa sesesencnn e ofoff ooothththererer isososofofoformrmrmss,s,

esesspepepecic ally FHL1AAA, in skkkeletaaal mumm sclelele wwwhhhat mmimight exxxpllaiaiainnn skkkeeletaall mmmuscllle hyyypepepertr ropphphyyy aananddd

cardiac dysfunction... ThThThe e findddininingggs of thee current stststududu y y haveveve iiimpmpm ortantt imimimplplp ications for diagnostic

evevalaluauatitionon, , , scscrereenenining,g,g, aandnd gggeneneteticic ccouounsnselelining g g ofof pppatatieientntss (a(a( lslso o cacarrrrieiersrs))) wiwithth mmyoyoy papap ththy y y ofof anan



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14

References:

1. Quadrelli R, Vaglio A, Reyno S, Lemes A, Salazar D, Lachman RS, Wilcox WR. Uruguay facio-cardio-musculo-skeletal syndrome: a novel X-linked recessive disorder. Am J Med Genet.2000;95:247-265.

2. Yourshaw M, Taylor SP, Rao AR, Martín MG, Nelson SF. Rich annotation of DNA sequencing variants by leveraging the Ensembl Variant Effect Predictor with plugins. Brief Bioinform. 2015;16:255-264.

3. McLaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics. 2010;26:2069-2070.

4. 1000 Genomes Project Consortium, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, et al. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012;491:56-65.

5. Kadrmas JL, Beckerle MC. The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol. 2004;5:920-931.

6. Loughna PT, Msaon P, Bayol S, Brownson C. The LIM-domain protein FHL1 (SLIM 1) exhibits functional regulation in skeletal muscle. Mol Cell Biol Res Commun. 2000;3:136-140.

7. Shathasivam T, Kislinger T, Gramolini AO. Genes, proteins and complexes: the multifaceted nature of FHL family proteins in diverse tissues. J Cell Mol Med. 2010;14:2702-2720.

8. Schessl J, Feldkirchner S, Kubny C, Schoser B. Reducing body myopathy and other FHL1-related muscular disorders. Semin Pediatr Neurol. 2011;18:257-263.

9. Cowling BS, Cottle DL, Wilding BR, D'Arcy CE, Mitchell CA, McGrath MJ. Four and a half LIM protein 1 gene mutations cause four distinct human myopathies: a comprehensive review of the clinical, histological and pathological features. Neuromuscul Disord. 2011;21:237-251.

10. Ng EK, Lee SM, Li HY, Ngai SM, Tsui SK, Waye MM, et al. Characterization of tissue-specific LIM domain protein (FHL1C) which is an alternatively spliced isoform of a human LIM-only protein (FHL1). J Cell Biochem. 2001;82:1-10.

11. Schoser B, Goebel HH, Janisch I, Quasthoff S, Rother J, Bergmann M, et al. Consequences of mutations within the C terminus of the FHL1 gene. Neurology. 2009;73:543-551.

12. Pen AE, Nyegaard M, Fang M, Jiang H, Christensen R, Mølgaard H, et al. A novel single nucleotide splice site mutation in FHL1 confirms an Emery-Dreifuss plus phenotype with pulmonary artery hypoplasia and facial dysmorphology. Eur J Med Genet. 2015;58:222-229.

5. Kadrmas JL, Beckerle MC. The LIM domain: from the cytoskeleton to the nucccleleeusuu ... NaNaNat t t ReReRev Mol Cell Biol. 2004;5:920-931.

6. LLLouououghghnaaa PPPTT, MMMsaon P, Bayol S, Brownson C. TTThhhe LIM-domain n n proteieieinnn FHL1 (SLIM 1) exexxhihihibits functctctiooonananal ll reeegugugulalalatititionoo iiin n n skskskelele etetetalalal mmusususclclc e. MoMoMol CeCeCellllll BBBioioi l ReReRes s s CoCoC mmmmmmun. 202020000000;3;3;3:11363636-1-1-1404040. .

7.7.. SSShahh thasivam TTT, Kisslinnnger T,T,T, Gramomm llinnni AO.O.O. Gennneees, prprproooteeinnns andndnd comppplexeeses: : : the mumumulttififaaacetededed natututurerere ooofff FHFHFHLLL fafafamimimilylyly ppprororotett inss s ininin dddiviviversesese tttisisissususueseses. JJJ CCCelele l ll MoMoMol ll MeMeMeddCCCCCC . 202020101010;1;1;14:4:4:272727020202-2-2-2727272000.

8. Schessl J, Feldkircrcrchnhnhnererer SSS, , , KuKuKubnbnbny y y C,C,C, SSSchchchosososererer BBB. ReReRedududuciiingngng bbbododody yy mymymyopopopatata hyhyhy aaandn other FHL1-eelalateted d mumuscsculularar ddisisorordedersrs.. SeSemimin n PePedidiatatr r NeNeururolol.. 20201111;1;1; 8:8:25257-7-26263.3.



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13. Poparic I, Schreibmayer W, Schoser B, Desoye G, Gorischek A, Miedl H, et al. Four and a half LIM protein 1C (FHL1C): a binding partner for voltage-gated potassium channel K(v1.5). PLoS One. 2011;6:e26524.

14. Windpassinger C, Schoser B, Straub V, Hochmeister S, Noor A, Lohberger B, et al. An X-linked myopathy with postural muscle atrophy and generalized hypertrophy, termed XMPMA, is caused by mutations in FHL1. Am J Hum Genet. 2008;82:88-99.

15. Knoblauch H, Greier C, Adams S, Budde B, Rudolph A, Zacharias U, et al. Contractures and hypertrophic cardiomyopathy in a novel FHL1 mutation. Ann Neurol. 2010;67:136-140.

16. Gueneau L, Bertrand AT, Jais JP, Salih MA, Stojkovic T, Wehnert M, et al. Mutations of the FHL1 gene cause Emery-Dreifuss muscular dystrophy. Am J Hum Genet. 2009;85:338-353.



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Table 1: Phenotype comparison among Uruguay, XMPMA and EDMD Plus and reducing body myopathy

Uruguay syndrome (1 family)

XMPMA (7 families)

EDMD Plus phenotype (1 family)

Reducing body myopathy (RBM)

Age onset Male: 1 - 5 yr, Female: mild, most unaffected

Male: 24 yrFemale: 44 yr

Male: as early as birth, Female: unaffected

Male: 5 yrFemale: 13 yr

Skeletal Scoliosis, toe and hip

dislocation, pes cavus, broad hands. No rigid spine

Rigid spine; scapular winging; contractures

Kyphosis, contractures, pectus deformity,

brachydactyly, wide feet

Rigid spine; scapular winging; contractures;

scoliosis

Cardiac Hypertrophy Hypertrophy Hypertrophy and arrhythmia Less common, dilated

Muscle Hypertrophy overall Atrophy of postural

muscles, hypertrophy of proximal upper limb

Proximal muscle weakness

Proximal muscle weakness

Dysmorphic facial

featuresYes No Yes No

Other No Respiratory involvement Pulmonary artery

hypoplasia, learning disability

Respiratory involvement

Death Young adult (~30) due to cardiac failure (untreated)

Adult (42 – 70) cardiac and respiratory failure

One age 16 (? arrhythmia)

Infantile and childhood respiratory failure

Reference 1 14 12 8

keletal Scoliosis, toe and hip

dislocation, pes cavus, broad hands. No rigid spine

Rigid spine; scapular winging; contractures

Kyphosis, contractures, pectus deformity,

brachydactyly, wide feet

RiRR gid sppine;; scapwiwiwingngnginining;g;g; cccononontrtrtraca tu

scscscolololioioiosisisisss

Carddiaiaaccc HyH pertrophy Hypertrororophphphyyy Hypertrophy yy and ararrhr ythmmmiaiaia Less common, dil

MuMM ssscle HHHypppertroophhhy y overrralalall lAAAtrophphp yyy of pooosttturaal l l

mumumuscless, hhhypertttrooophy oofff prroximmmaall uppeeer limmbbb

Prroxoxoxiimal mmmussscle wwweaknessss

PrPP oxxximmmala mmmususscweweweaka neeess

smorphphhicicic facial eatures

YeYeYesss NoNo YeYeYesss No

Pulmonary artery



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17

Figure Legends:

Figure 1: Updated pedigree with segregation analysis of available family members.

Figure 2: A. Isoforms of FHL1. B. Splice site changes of exon 6 in Uruguay syndrome,

XMPMA and EDMD plus patients.

Figure 3: A. Western blot for FHL1 A. Different dilutions (1:5 and 1:2) and different amounts

(10uL and 5uL) of antibody to FHL1A and duplicates of Uruguay patient (A and B) were used.

In all patient samples there is no normal FHL1 band at 37kDa, but a minor degraded band at ~ 28

kDa. As a loading cytoskeletal control marker at the bottom of the western blot, caveolin-3 was

used with a band at 21 kDa. B. Immunohistochemistry for FHL1A. Immunohistochemistry was

performed in muscles from Uruguay syndrome patient, healthy controls, and a Becker muscular

dystrophy patient. In the Uruguay syndrome patient the FHL1 antibody shows no staining

compared to the normal membrane staining in the muscles of both controls and the Becker

muscular dystrophy patient. All internal non-membrane bound staining is an artifact. Bars

adjusted to 50μm.

Figure 4: Expression of FHL1 isoforms in myoblasts. The expression of all FHL1 isoforms

(FHL1A, FHL1B and FHL1C) was tested in myoblasts from healthy individuals (control), from

patients with a c.688+1G>A mutation (donor splice site mutation) and the Uruguay patient. In

general, quantification experiments were performed in triplicates for each sample, except for the

expression of FHL1 A/B/C of the sample with the c.688+1G>A mutation, where due to a

n all patient samples there is no normal FHL1 band at 37kDa, but a minor degradadadededed bbbananand d d atatat ~ 28

kDa. AAss a a loaadingngg cytoskeletal control marker at thhee bottom of the westerern n blot, caveolin-3 was

usususeddd with a bababandndnd aaat 21211 kkkDaDaDa... B.B.. IIImmmmmmununnohohohistototochchchemiistttry y fofofor rr FHFHFHL111A.A.A. IIImmmmmmunununohhisisistototochchchemememisisistrtrtry y y wawawas ss

pepeerffforoo med in muuuscccles frrrom UrUrUruugu uay y y sysyynddromememe patttieeent,,, heheheallthhhy cooonnntrols,,, aaand d d a a Beckkkererr mmmuususculllarrr

dystrophy patient. In n n thththee Uruguguguauauay y syynddrome pppatieeentntn the FHLHLHL1 antiboodydydy ssshows no staining

cocompmpparareded tto o ththee nonormrmalal mmemembrbranane e ststaiaininingngg iin n ththee mumuscscleles s ofof bbototh h cocontntrorolsls aandnd tthehe BBececkeker r



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18

technical error one dataset was excluded. The error bars in the three panels display the calculated

mean, maximum and minimum expression levels presented as RQ, RQmin and RQmax. RQmax

and RQmin are based on the 95% confidence interval of Ct.



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A



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B



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WilcoxBeichler, Stanley F. Nelson, Gudrun Schappacher-Tilp, Christian Windpassinger and William R.

Yuan Xue, Benedikt Schoser, Aliz R. Rao, Roberto Quadrelli, Alicia Vaglio, Verena Rupp, Christinean X-Linked Disorder with Skeletal Muscle Hypertrophy and Premature Cardiac Death

that Causes Uruguay Syndrome,FHL1Exome Sequencing Identified a Splice Site Mutation in

Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2016 American Heart Association, Inc. All rights reserved.

TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

published online March 1, 2016;Circ Cardiovasc Genet.

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The online version of this article, along with updated information and services, is located on the

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Exon spanning primer Sequence

FHL1_RT2_E3-4f 5’-GTTCTGTGCCAACACCTGTG

FHL1_RT2_E3-4r 5-‘CAGGTGTCATGCCAGAAGC

FHL1_RT2_E5-6f 5’-GACTTGCCATGAGACCAAGTT

FHL1_RT2_E5-6r 5’-GGGCTGATCCTGGTAAGTGA

FHL1_RT1_E5-8f 5’-GTGCAACAAGGGTTTGGTAAAG

FHL1_RT1_E5-8r 5’-CGGAGCATTTTTTGCAGTG

GAPDH_f 5’-CCACTCCTCCACCTTTGAC

GAPDH_r 5’- ACCCTGTTGCTGTAGCCA

Supplementary table 1. Primer sequences for FHL1 isoforms

SUPPLEMENTAL MATERIAL

Variant ID Consequence on protein Gene

Amino acid

change

SIFT

prediction

PolyPhen

prediction

Condel

prediction Conservation*

CADD

score†

Protein evidence‡

Expression

in heart

muscle

myocytes§

Expression

in skeletal

muscle

myocytes§

Bin| |

X:129147728 C>T non_synonymous_codon BCORL1 T327I deleterious unknown 4.27 19.46 1:Evidence at protein level Medium High 2

X:132091263 G>A non_synonymous_codon HS6ST2 P174S tolerated probably_damaging deleterious 4.15 23.4 2:Evidence at transcript level Negative Negative 9

X:132092621 G>A non_synonymous_codon HS6ST2 P4S tolerated benign neutral 4.03 14.2 2:Evidence at transcript level Negative Negative 9

X:134950091 C>G non_synonymous_codon CT45A2 M52I tolerated benign neutral 1.36 4.324 2:Evidence at transcript level 3

X:135290612 A>G splice_acceptor_variant FHL1 4.17 23.2 1:Evidence at protein level Medium High 1

X:135313837 T>C rs148588752 non_synonymous_codon MAP7D3 K427E tolerated benign neutral -3.77 0.001 1:Evidence at protein level Weak Moderate 6

X:135477570 C>T rs137972430 non_synonymous_codon GPR112 S2454L deleterious benign neutral 2.8 27.1 2:Evidence at transcript level Strong Moderate 7

X:135957716 A>G rs142284545 non_synonymous_codon RBMX M82T tolerated benign neutral -2.85 0.589 2:Evidence at transcript level 7

X:148674677 C>T non_synonymous_codon HSFX2 A283T tolerated benign neutral -2.7 0.026 4

X:148674706 G>A non_synonymous_codon HSFX2 P273L tolerated benign neutral 0.751 21.9 4

Supplementary Table 1. Rare variants shared between cases. Annotations are displayed for the transcript on which given variant has the most deleterious consequence. The splice variant selected for confirmation is highlighted in bold.

* Scores were obtained using GERP. High numbers indicate variants that are conserved between species.

† Scores are PHRED-like scaled C-scores from the Combined Annotation-Dependent Depletion (CADD) method, that may be used as a measure for deleteriousness. A score >=20 indicates variants predicted to be in the top 1% of the most

deleterious possible substitutions in the human genome.

‡ Obtained using the Uniprot database, indicating whether there is clear experimental evidence for the existence of the protein or transcript.

§ Based on data in the Human Protein Atlas.

| | Bins defined as the following. 1-4: Variant absent from public datasets (1000 Genomes Project, NIEHS, NHLBI EVS, dbsnp135); 5-8: Variant observed in public datasets; 1,5: Protein evidence(PE)=1 and high impact consequence on protein;

2,6: PE=1 and non-synonymous variant; 3,7: PE=2; 4,8: PE missing; 9: Not expressed in heart muscle or skeletal myocytes

Supplementary Table 2. Rare variants shared between cases. Annotations are displayed for the transcript on which given variant has the most deleterious consequence. The splice variant selected for confirmation is highlighted in bold.

Supplementary FHL1 blotThe FHL1 band at 37kDa was delectable in all controls (Co1-Co4) and samples from Becker (BMD) and Duchenne muscular dystrophy (DMD) at normal intensity. Note that the band of 37kDa was slightly weaker in both BMD samples.

exome sequencing identified a splice site mutation in fhl1...

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