Exome Sequencing Identifies GNB4 Mutations As a Cause of Dominant

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Exome Sequencing Identiﬁes GNB4 Mutations as a Cause of Dominant Intermediate Charcot-Marie-Tooth Disease

Bing-Wen Soong,1,2,3,4,13 Yen-Hua Huang,5,6,13 Pei-Chien Tsai,3 Chien-Chang Huang,3,7 Hung-Chuan Pan,8,9 Yi-Chun Lu,1 Hsin-Ju Chien,1 Tze-Tze Liu,10 Ming-Hong Chang,2,11 Kon-Ping Lin,1,2,3 Pang-Hsien Tu,12 Lung-Sen Kao,3,7,10,* and Yi-Chung Lee1,2,3,*

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identiﬁed as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28–q29. Subse- quent exome sequencing of two affected ﬁrst cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding

guanine-nucleotide-binding protein subunit beta-4 (Gb4), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in

one individual. Immunohistochemistry studies revealed that Gb4 was abundant in the axons and Schwann cells of peripheral nerves > and that expression of Gb4 was signiﬁcantly reduced in the sural nerve of the two individuals carrying the c.158G A (p.Gly53Asp) mutation. In vitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-

coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gb4. This study identiﬁes GNB4 mutations as a cause of CMT

and highlights the importance of Gb4-related GPCR signaling in peripheral-nerve function in humans.

Charcot-Marie-Tooth disease (CMT) is a group of inherited being asymptomatic to being wheelchair-bound. The neuropathies characterized by progressive distal muscle median motor NCVs ranged from 16.5 to 45.7 m/s. Men atrophy, distal sensory loss, depressed tendon reflexes, tended to be more severely affected (Table 1). Sural nerve and foot deformities; they can be categorized into either biopsies of two affected individuals detected loss of the demyelinating form or the axonal form by their elec- myelinated fibers and also demyelinating changes as trophysiological and pathological features.1,2 Clinically, evidenced by multiple onion-bulb formations. Written median motor nerve conduction velocity (NCV) is the informed consent was obtained from all subjects, and the most frequent parameter (with a cutoff value of 38 m/s) study protocol was approved by the institutional review used for distinguishing between demyelinating and axonal board at Taipei Veterans General Hospital, Taipei, Taiwan. CMT.3 Mutations in approximately 45 genes have been Genomic DNA was isolated from peripheral-blood leuko- identified as being associated with CMT, but these account cytes according to standard protocol. for fewer than 70% of all CMT cases.1,2 Within the CMTs, About 165,000 exonic regions from the genomic DNA of dominant intermediate CMT (DI-CMT) is a rare group the two affected first cousins of family 1 (IV-1 and IV-4 in that shows autosomal-dominant inheritance and variable Figure 1A) were captured and enriched with the Agilent NCVs, which can look like either demyelinating or axonal SureSelect Human All Exon v.2 kit. The DNA-sequencing types of CMT within a pedigree.3–6 Previously, we charac- libraries were prepared with an Illumina Paired-End DNA terized a three-generation Chinese family afflicted with Sample Preparation kit. A total of 100 bp paired-end reads a DI-CMT and mapped the locus of the candidate gene to were obtained by sequencing on an Illumina Genome chromosomal region 3q28–q29.7 In this study, 13 family Analyzer GAIIx. All sequence reads were mapped to the members, including six affected individuals, five unaf- human reference genome (GRCh37 patch 2) with the fected individuals, and two married-in spouses, were Burrows-Wheeler Aligner.8 Removal of PCR duplicates enrolled (Figure 1A). Disease status was determined by and variant calling were performed with SAMtools/ both the clinical manifestations and electrophysiological BCFTools.9 Only the variants with Phred-like scores of at evidence.3 In brief, the age of onset ranged from 10 to 45 least 30.0 were collected for the investigation of candidate years, and individuals ranged in clinical severity from variants of high confidence. On average, 6 Gb of sequence

1Department of Neurology, Taipei Veterans General Hospital, Taipei 11217, Taiwan; 2Department of Neurology, National Yang-Ming University School of Medicine, Taipei 11221, Taiwan; 3Brain Research Center, National Yang-Ming University, Taipei 11221, Taiwan; 4Institute of Neuroscience, National Yang- Ming University, Taipei 11221, Taiwan; 5Department of Biochemistry, National Yang-Ming University School of Medicine, Taipei 11221, Taiwan; 6Center for Systems and Synthetic Biology, National Yang-Ming University, Taipei 11221, Taiwan; 7Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan; 8Faculty of Medicine, National Yang-Ming University, Taipei 11221, Taiwan; 9Department of Neuro- surgery, Taichung Veterans General Hospital, Taichung 40705, Taiwan; 10Genome Research Center, National Yang-Ming University, Taipei 11221, Taiwan; 11Section of Neurology, Taichung Veterans General Hospital, Taichung 40705, Taiwan; 12Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan 13These authors contributed equally to this work *Correspondence: [email protected] (Y.-C.L.), [email protected] (L.-S.K.) http://dx.doi.org/10.1016/j.ajhg.2013.01.014. Ó2013 by The American Society of Human Genetics. All rights reserved.

422 The American Journal of Human Genetics 92, 422–430, March 7, 2013 Figure 1. Pedigrees and Electrophero- grams of the GNB4 Mutations (A and B) Pedigree structure of families 1 (A) and 2 (B). The probands are denoted by an arrow. The squares and circles denote males and females, respectively, and the diagonal black lines represent deceased individuals. Filled symbols represent affected members with neuropathy, and open symbols indicate unaffected individuals. The numbers in diamonds indicate the number of unaffected siblings or children. The gender and birth order have been partially hidden for the sake of conﬁdentiality. ‘‘M’’ represents the GNB4 mutant allele, ‘‘W’’ stands for the wild- type allele, and asterisks denote members who underwent whole-exome sequencing. (C) The electropherograms of the heterozygous GNB4 mutations, c.158G>A (p.Gly53Asp) and c.265A>G (p.Lys89Glu). (D) RT-PCR analysis of the RNA isolated from the leukocytes of a normal control and the individual carrying GNB4 c.265A>G with the use of primers speciﬁc to GNB4 exons 5 and 6. Both samples revealed a single band at the same location (218 bp), indicating that GNB4 mRNA from the affected individual was not differ- entially spliced. (E) The cDNA electropherograms of the GNB4 exon 5 and 6 junction of a normal control and the affected individual carrying GNB4 c.265A>G reveal similar splicing patterns.

mutation, c.265A>G (p.Lys89Glu) (Figures 1B and 1C) in the heterozygous form, was identiﬁed. Neither the GNB4 c.158G>A mutation nor was generated from each individual, resulting in a coverage the c.265A>G mutation was present in the Exome depth of 833 per base per targeted region. After analysis Variant Server of the National Heart, Lung, and Blood Insti- (Table S1, available online), 64 heterozygous coding vari- tute (NHLBI) Exome Sequencing Project or in 1,920 ethni- ants were found in both individuals; these variants were cally matched control chromosomes. Gly53 and Lys89 are not present in dbSNP, the 1000 Genomes Project,10 or highly conserved residues in Gb4(Figure S1). SNPs&GO,11 the in-house exome data from ten non-CMT ethnic Panther,12 and Polyphen213 predicted both mutations to 14 controls. Targeted sequencing of eleven other members be deleterious to Gb4 functioning (Table S3). Because of family 1 revealed that only the c.158G>A (p.Gly53Asp) the c.265A>G mutation occurs at the nucleotide adjacent variant in GNB4 (MIM 610863; RefSeq accession number to the exon-intron boundary (Figure 1C), we analyzed the NM_021629.3), encoding guanine-nucleotide-binding mRNA from leukocytes of the individual carrying this protein (G protein) subunit beta-4 (Gb4), perfectly segre- mutation by RT-PCR and nucleotide sequencing. No gated with the CMT phenotype (Figures 1A and 1C). This splicing alteration was found (Figures 1D and 1E). We variant was also located within the chromosomal region also utilized a minigene assay to demonstrate that the 3q28-q29, which was the previously mapped locus for GNB4 c.265A>G mutation did not change the mRNA this type of CMT. splicing pattern (Figure S2). Because the CMT individuals To support that mutations in GNB4 cause CMT and to in this study were selected from 251 pedigrees affected by investigate the frequency of GNB4 mutations in our CMT CMT after the cases with clear genetic diagnosis were population, we further sequenced all nine coding exons excluded,15 the frequency of GNB4 mutations in our of GNB4 in another 88 unrelated individuals with molecu- CMT population was approximately 0.8% (2/251). larly unassigned CMT (Table S2); these individuals The GNB4 c.265A>G (p.Lys89Glu) mutation was found included 66 with the demyelinating variant and 22 with in a 9-year-old girl who had presented with slowly progres- the axonal variant. Among them, one additional de novo sive weakness of her distal lower limbs since the age of

The American Journal of Human Genetics 92, 422–430, March 7, 2013 423 Table 1. Clinical Features of the Individuals with GNB4 Mutations Family 2 (c.265A>G Family 1 (c.158G>A [p.Gly53Asp]) [p.Lys89Glu])

III-2 III-3 IV-1 IV-2 IV-3 IV-4 II-1

Age of onset (years) 13 45 10 13 17 22 5

Age at exam (years) 49 48 23 18 17 22 9

Gender male female male male female female female

Upper-limb weakness (MRC) distal: 4 normal normal normal normal normal normal

Lower-limb weakness (MRC) distal: 2 distal: 4- to 4 distal: 4- to 4 distal: 4 distal: 4þ distal: 4þ distal: 4

Upper-limb muscle atrophy distal: moderate normal distal: mild normal normal normal normal

Lower-limb muscle atrophy distal: severe distal: mild distal: severe distal: mild distal: mild distal: mild distal: moderate

Knee and ankle DTRs absent absent absent absent absent reduced or absent absent

Gait bound in steppage steppage steppage normal normal steppage wheelchair

Pinprick sensation reduced mildly reduced mildly reduced mildly reduced normal normal mildly reduced

Vibration reduced mildly reduced mildly reduced mildly reduced normal normal mildly reduced

Median nerve MNCV (m/s) 25.2 35.4 16.5 28.6 44.2 45.7 20

Median nerve cMAP (mV) 7.7 8.3 3.9 3.2 9.3 5.9 2.6

Sural nerve SNAP NR NR NR NR NR 7 NR

Sural nerve biopsy demyelination demyelination ND ND ND ND ND

The following abbreviations are used: MRC, Medical Research Council scale; DTR, deep tendon reﬂex; MNCV, motor nerve conduction velocity; cMAP, compound motor action potential; SNAP, sensory nerve action potential; NR, not recordable; and ND, not done.

5 years. She had started to walk at a normal age. Physical Gbg dimer each then transduce signals to a wide range of examination at age 9 years revealed high-arched feet, effectors, including adenylyl cyclases, phospholipases, hammer toes, atrophy and weakness of the intrinsic ion channels, phosphodiesterases, and many more pro- muscles of the feet (score 4/5 on the Medical Research teins,17,18 thereby regulating various intracellular signaling Council Scale), impaired dorsiflexion of the feet (score pathways and cellular functions. As a result of this wide 4/5), generalized areflexia, and mildly diminished sensa- range of G protein signaling activities, GNB4, which tion for all modalities in regions distal to the ankles despite encodes Gb4, is likely to be important to a number of a lack of sensory complaints. Nerve conduction studies cellular signal-transduction systems. demonstrated the presence of demyelinating sensorimotor To understand the location of Gb4 in peripheral nerves, polyneuropathy with axonal loss. The median and tibial we analyzed the sural nerves of one neurologically normal motor NCVs were 20 and 15 m/s, respectively. Neither control and one individual (III:3 in Figure 1A) with the parent had the CMT phenotype or the GNB4 c.265A>G c.158G>A (p.Gly53Asp) GNB4 mutation by double immu- mutation (Figure 1B). nofluorescence staining. Gb4 was found to be colocalized G proteins are heterotrimeric and are composed of a, b, with neurofilament heavy chain and S100 (Figure 2 and and g subunits; they are important to cellular signal Table S4), indicating that Gb4 is expressed in both axons transduction. They transmit extracellular signals into cells and Schwann cells. Intriguingly, some myelinated fibers by acting in concert with different G-protein-coupled of the normal control had a target-like appearance because receptors (GPCRs); these GPCRs can be activated by of the presence of dense Gb4 staining in the axons and the many different signal factors, such as neurotransmitters, outer rim of the myelin, but not in the compacted myelin hormones, and cytokines.16 In humans, there are 16 Ga sheath (Figure 2). Onion-bulb formations in the sural subunits, 5 Gb subunits, 12 Gg subunits, and a number nerve of individual III:3 showed a ‘‘rosette’’ pattern with of splice variants, which thus contribute to a large diversity Gb4 staining in the axons and the cytoplasm of sur- of G protein heterotrimers.17 In the inactive state, the rounding Schwann cells (Figure 2). guanosine-diphosphate (GDP)-bound Ga is associated We next compared Gb4 expression in the sural nerve with the Gbg dimer. When a GPCR is activated by a ligand, samples from two individuals (III:2 and III:3) with the it catalyzes the G protein to exchange the GDP for guano- GNB4 c.158G>A (p.Gly53Asp) mutation, one individual sine triphosphate (GTP) on Ga, which results in the disso- with CMT1A, and one neurologically normal control ciation of the Ga from the Gbg.16 The Ga-GTP and released by immunohistochemistry (IHC). In addition to staining

424 The American Journal of Human Genetics 92, 422–430, March 7, 2013 crush injury, or a sham operation. The nerve tissue 10 mm

in length and distal to the injured site was analyzed for Gb4 expression by immunoblotting and IHC. Compared with

Gb4 expression in the sham-operated group, the expres-

sion of Gb4 appeared to increase in the nerve conditioning group and decrease in the nerve transection group (Fig- ure S3). These ﬁndings suggest that GNB4 plays a role in peripheral nerve regeneration and support its importance in peripheral nerve functioning. To investigate the effect of the GNB4 mutations on

protein stability and G-protein-coupled signaling by Gb4,

we constructed Gb4 expression plasmids to allow further in vitro analysis. The coding region of GNB4 from a human GNB4 cDNA clone (Invitrogen; GenBank accession þ number AF300648.1) was subcloned into pcDNA3.1 (Invitrogen). Mutations c.158G>A (p.Gly53Asp) and c.265A>G (p.Lys89Glu) were introduced by site-directed mutagenesis according to the Quick-Change method (Stratagene) (Table S5). pECFP-C1 was purchased from

Clontech. A human PLCb2 expression clone, pCMV5-

PLCb2, was a kind gift from E.M. Ross (Southwestern Medical Center, University of Texas, TX, USA). All con- structs were verified by DNA sequencing. In order to carry out a protein-stability assay, we incubated human embryonic kidney (HEK) 293T cells maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) containing 10% fetal bovine serum (FBS; Invitrogen) at 37 C and in 5% CO2 and transfected them with plasmids expressing wild-type, p.Gly53Asp, or b Figure 2. G 4 Expression in Both Axons and Schwann Cells of p.Lys89Glu forms of Gb4 by using calcium-phosphate Peripheral Nerves 19 Double immunostaining of Gb4 with neurofilament heavy chain precipitation. At 48 hr after transfection, the cells were (NF-H) (A) or S100 (B) with sural nerve samples from a neurologi- treated with 100 mg/ml cyclohexamide for 0, 2, 4, 8, or cally normal control and family 1 individual III:3, who harbors the 24 hr. Total-protein cell extracts were analyzed by immu- GNB4 c.158G>A (p.Gly53Asp) mutation. The colocalization of noblotting (40 mg of protein was loaded from each cell Gb4 with NF-H and S100 indicates that Gb4 is expressed in both axons and Schwann cells. Some myelinated fibers of the normal lysate). The immunoblot signals were used for comparing control have a target-like appearance as a result of dense Gb4 stain- the relative degradation profiles between the Gb4 variants ing in the axons and the outer rim of the myelin, but not in the and actin. Representative graphs and quantification ob- compacted myelin sheath (arrows). Onion-bulb formations in tained from six independent experiments are shown in the sural nerve of individual III:3 have a ‘‘rosette’’ pattern with Gb4 staining in the axons and the cytoplasm of surrounding Figure S4. These results show that the degradation rates Schwann cells (arrowheads). The scale bars represent 10 um. of the two altered forms of Gb4 were not significantly different from that of their wild-type counterpart, suggest-

Gb4, we also doubly stained the cell nuclei with TDP-43 ing that these alterations did not affect protein stability. antibody as a reference (Table S4). Immunostaining re- We next investigated the functional impact of the Gb4 vealed that Gb4 was abundantly expressed in the axons alterations on GPCR signaling. In this functional study, and Schwann cells of the sural nerve of the normal control we used bradykinin to activate the B2-receptor-coupled

(Figure 3A). Some myelinated fibers had a target-like Gqa signaling pathway in a PLCb2-overexpressing COS-7 20,21 appearance (Figure 3A). Onion-bulb formations in the cell model. Activation of PLCb2 by Gqa signaling leads sural nerve of the CMT1A control were also densely stained to hydrolysis of phosphatidylinositol 4,5-bisphosphate and with Gb4 antibody (Figure 3B). In contrast, Gb4 staining the formation of the second messengers inositol trisphos- was significantly weaker in the sural nerves of the two indi- phate (IP3) and diacylglycerol, which is followed by IP3- 2þ viduals with the GNB4 mutation than in those of the induced intracellular Ca release. Thus, IP3 production normal control and the individual with CMT1A (Figures and cytosolic calcium changes were used to reflect GPCR 3C and 3D). signaling activity. In brief, COS-7 cells were maintained in To test the hypothesis that GNB4 is important for DMEM containing 10% FBS at 37 C and in 5% CO2; peripheral nerve functioning, we investigated Gb4 expres- LipofectAmine-2000 (Invitrogen) was used for transfecting sion in the sciatic nerves of the rats 3 days after sciatic these cells at a 1:1:1 ratio with plasmids expressing ECFP,ex- nerve transection, nerve conditioning with a 20 s transient pressing PLCb2, and expressing wild-type, p.Gly53Asp, or

The American Journal of Human Genetics 92, 422–430, March 7, 2013 425 b Figure 3. G 4 Immunohistochemistry and Toluidine-Blue Staining of Sural Nerves Double immunostaining of Gb4 (red) and TDP-43 (dark blue) in sural nerve samples from a neurologically normal control (A), a disease control with CMT type 1A (CMT1A) (B), and family 1 individuals III:2 (C) and III:3 (D), who harbor the GNB4 c.158G>A (p.Gly53Asp) mutation. Some myelinated ﬁbers in the normal control feature a target-like appearance (A, arrows). The Gb4 staining of onion- bulb formations appears weaker in the two subjects with the GNB4 mutation (C and D, arrowheads) than in the individual with CMT1A (B, arrowheads). The sural nerves of the two individuals with the GNB4 mutation have obviously weaker Gb4 staining than do those in the normal control and the individual with CMT1A. The cell nuclei were stained with TDP-43 antibody as a reference. The toluidine- blue staining of sural nerve samples of individuals III:2 and III:3 from family 1 reveals a loss of myelinated ﬁbers and a presence of multiple onion-bulb formations (C and D), associated with a demyelinating change. The scale bars represent 10 um.

impaired, that is, they showed

reduced bradykinin-induced PLCb2 activation, namely an inhibition of p.Lys89Glu forms of Gb4 or empty vectors. Forty-eight IP3 production and a lower increase in cytosolic calcium. hours after transfection, the cells were harvested for subse- Compared to control cells, PLCb2-overexpressing cells quent second-messenger analysis. To measure IP3 produc- showed a bradykinin-induced increase in IP3 production tion, we subcultured the cells into a 96-well plate at a density by 25% (25.7% 5 6.2%, n ¼ 3). When cells coexpressed 4 of 2 3 10 cells per well with 20 ml PBS. The cells were then wild-type Gb4 and PLCb2, their IP3 generation was found stimulated with 100 nM bradykinin for 1 min and lysed to be about 3.5-fold higher than that of the PLCb2- with 0.2 N perchloric acid. Intracellular IP3 production overexpressing cells (87.9 5 11.1% versus 25.7% 5 was then measured with a HitHunter IP3 Fluorescence 6.2%). However, compared to the control, the p.Gly53Asp

Polarization Assay Kit (DiscoveRx) as outlined in the manu- and p.Lys89Glu altered forms of Gb4 reduced bradykinin- 5 5 facturer’s manual. To measure cytosolic calcium changes, induced IP3 production by 42.1% 2.8% and 45.7% we incubated cells on coverslips with 5 mM of the calcium 6.0%, respectively (Figure 4A). Similar results were ob- 2þ indicator dye fura-2 AM (Molecular Probes) in loading served when the bradykinin-induced [Ca ]i increases buffer (LB) for 45 min at 37C and then washed them were monitored. Bradykinin induced a similar increase in 2þ with LB. Cells coexpressing ECFP with a similar expression [Ca ]i when the controls, the PLCb2-overexpressing cells, level (mean pixel intensity ¼ 526.6 5 32.8 AU; n ¼ 148) and the cells coexpressing PLCb2 and wild-type Gb4 were were selected for the subsequent measurements. These cells compared. However, when the cells coexpressing either were stimulated with a pulse of 100 nM bradykinin for 10 s PLCb2 and p.Gly53Asp Gb4 or PLCb2 and p.Lys89Glu while the fluorescence of fura-2 was simultaneously moni- Gb4 were examined, there were no significant changes in 2þ tored under an inverted microscope (IX-71, Olympus [Ca ]i upon bradykinin stimulation (Figures 4B and 4C). Optical) coupled with a monochromator (Polychrome IV, These findings indicate that the mutations in GNB4 cause TILL Photonics, Gra¨felfing, Germany) with a 403 oil- a defect in the GPCR signaling cascade and impair the immersion objective (UApo/340 403/1.35 numeric aper- activation of PLCb2. ture oil objective, Olympus Optical).22 Use of the 340/ Utilizing exome sequencing, bioinformatic analysis, and 380 nm excitation ratio with fura-2 allowed measurements mutational analysis, we have identified two mutations that 2þ 23,24 of the intracellular calcium concentration ([Ca ]i). affect Gb4-encoding GNB4 and are present in subjects The study revealed that the function of both the with CMT. Both GNB4 mutations alter the evolutionarily p.Gly53Asp and p.Lys89Glu altered forms of Gb4 was conserved amino acid residues, are absent from the NHLBI

426 The American Journal of Human Genetics 92, 422–430, March 7, 2013 Exome Variant Server, and do not occur in 1,920 ethnic control chromosomes. Immunohistochemical studies re-

vealed that Gb4 is abundantly expressed in peripheral nerves, including both axons and Schwann cells. In vitro studies demonstrated that both GNB4 mutations impair bradykinin-induced GPCR signaling. Our ﬁndings provide strong genetic and functional evidence supporting that mutations in GNB4 are the cause of this type of CMT. According to the tissue expression databases of 25 26 UniGene and GeneNote (now retired), Gb4 is widely expressed in many human tissues, including the spinal

cord and peripheral nerves. Gb4 has also been found to be expressed in peripheral nerve myelin by proteome anal- yses.27 We have been able to consistently demonstrate that

Gb4 is readily detected in the axons and Schwann cells of

sural nerves, which indicates that the Gb4 signal pathway is essential for peripheral nerve functioning. Decreased

Gb4 immunostaining in the sural nerves of the two CMT individuals with GNB4 mutations further highlights the

important role of Gb4 dysfunction in the pathogenesis of this hereditary neuropathy. Our functional studies have shown that both the

p.Gly53Asp and p.Lys89Glu Gb4 alterations impair GPCR signaling pathways via a dominant-negative effect. When

compared to cells expressing PLCb2 alone, cells coexpressing PLCb2 and p.Gly53Asp or p.Lys89Glu altered Gb4

showed a signiﬁcantly reduced IP3 production and an absence of cytosolic calcium change when stimulated with bradykinin. This phenomenon suggests that both

altered forms of Gb4 have a negative effect on the endoge-

nous wild-type Gb4 molecules already present in the cells and that this affects GPCR signaling. Gly53 and Lys89 are highly conserved across the Gb protein family (in-

cluding Gb1–Gb5) across the various kingdoms (including mammals, fungi, nematode, and amoeba). This suggests that they both most likely play critical roles in GPCR Figure 4. Inhibition of the GPCR Signaling Results from GNB4 signaling.28 Mutations The functional defect caused by the two mutations (A) The p.Gly53Asp and p.Lys89Glu altered forms of Gb had an 4 appears to be related to their roles in the specific binding inhibition of inositol trisphosphate (IP3) production upon bradykinin stimulation. COS-7 cells were transfected with pcDNA3.1 of Ga to the Gbg dimer. On the basis of the three- (control, first bar on the left), pcDNA3.1-PLCb2 alone, or dimensional (3D) structure of the native heterotrimer, pcDNA3.1-PLCb and pcDNA3.1-Gb or altered forms of Gb . 2 4 4 Gia1b1g2, the first two WD40 repeats of Gb1 interact with The IP3 generated upon stimulation with 100 nM bradykinin for the N-terminal helix of Gia1, and it has been suggested 1 min was then measured. The IP3 production level was normal- ized against the control as 100%. The data shown are the that this interaction confers a certain degree of specificity 5 29 mean SEM from three independent transfections. to Ga-Gbg complex formation. Gb4 shares 96% amino þ (B) Absence of changes in intracellular calcium ([Ca2 ] ) upon 30 i acid sequence homology with Gb1. Gly53 and Lys89 bradykinin stimulation in the cells expressing altered forms of are located within the first and second WD40 repeats, Gb4. COS-7 cells were transfected with pcDNA3.1 (control), respectively, of the b propeller, and according to high-reso- pcDNA3.1-PLCb2 alone, or pcDNA3.1-PLCb2 and pcDNA3.1-Gb4 or altered forms of Gb4 in conjunction with pECFP-C1. Changes lution structure analysis, Gly53 contacts one residue and in the F340/F380 ratio of fura-2 upon 100 nM bradykinin stimula- Lys89 contacts multiple residues within the N-terminal tion were monitored by a fluorescence microscope. Bradykinin 29 2þ helix of Gia1 (Figure S5). Therefore, it is likely that the stimulation started at 30 s and lasted for 10 s. The [Ca ]i changes interaction between Gb and Ga, but not the interaction are shown as the F340/F380 ratio of fura-2 fluorescence. 4 2þ (C) Maximal [Ca ]i changes upon bradykinin stimulation. The between Gb4 and Gg, is affected in both the p.Gly53Asp 2þ maximal [Ca ]i changes were obtained by subtraction of the aver- and p.Lys89Glu Gb4 altered proteins. Although the dimer- aged fluorescence ratio in the 10 s before stimulation from the ization of Gb and Gg is obligatory in nature,31 the interac- maximal fluorescence ratio. The data shown are the mean 5 SEM from 26–34 cells for each group (148 cells in total). The cells tion between Gg and the altered forms of Gb4 might render were obtained from at least three independent transfections. a decrease in the functional wild-type Gb4-Gg dimer and

The American Journal of Human Genetics 92, 422–430, March 7, 2013 427 thus exert a dominant-negative effect that abolishes rele- negative effect. Such a toxic function could result in a vant GPCR signaling. situation where cells expressing the most altered form of

All of the individuals carrying the GNB4 c.158G>A Gb4 are likely to die ﬁrst. In such a circumstance, the re- (p.Gly53Asp) or c.265A>G (p.Lys89Glu) mutation feature maining cells in the sural nerves of these individuals would a typical CMT phenotype that involves distal motor and be those with relatively lower Gb4 expression. This specu- sensory function; however, the subject carrying the lation is supported by the lower cell and nerve-ﬁber densi- c.265A>G (p.Lys89Glu) mutation had an earlier age of ties in the sural nerve samples of the two individuals with onset, indicating that she was more severely affected. a GNB4 mutation than in those in the control samples The c.265A>G (p.Lys89Glu) mutation seemed to affect (Figure 3).

Gb4 functioning more than the c.158G>A (p.Gly53Asp) We have also screened for mutations in the genes encod- mutation in our small subject group. This is supported by ing the Gg subunits that are known to be expressed in the the 3D structure of the heterotrimeric Gia1b1g2. Whereas peripheral nerves; these genes included GNG3 (MIM

Gly53 of Gb1 only interacts with Leu23 of Gia via van 608941), GNG4 (MIM 604388), GNG7 (MIM 604430), der Waals interactions, Lys89 interacts with multiple resi- GNG8, and GNG10 (MIM 604389) across 87 molecularly dues, namely Gia via hydrogen bond with Ser16, via unassigned and unrelated CMT individuals. So far, we a salt bridge with Asp20, and via van der Waals interactions have not identiﬁed any relevant mutation in them (data with Ile19 and Leu23 (Figure S5).29 Thus, Lys89 might be not shown). The possibility that other proteins that more important than Gly53 in terms of the association interact with Gb4 might also be pivotal to the etiology of between subunits Ga and Gb, and this might therefore some of these hereditary neuropathies cannot be ruled out. cause the c.265A>G (p.Lys89Glu) mutation to produce a In conclusion, our ﬁndings demonstrate that mutations more severe disease phenotype. in GNB4 cause CMT, and this emphasizes the importance

The diagnosis of DI-CMT in family 1 was based on the of Gb4-related GPCR signaling to peripheral nerve func- NCVs and the sural nerve biopsies of subjects III:2 and tioning in humans. III:3, who had slow NCVs consistent mainly with demyelinating neuropathy. Because NCV cannot faith- Supplemental Data fully reflect the underlying pathological changes, the full Supplemental Data include five figures and five tables and can be pathological spectrum of Gb4-related neuropathy is yet found with this article online at http://www.cell.com/AJHG/. to be delineated. Given that Gb4 is highly expressed in both axons and Schwann cells, mutations in GNB4 might result in myelinopathy and/or axonopathy; this might Acknowledgments partially explain the phenotypic heterogeneity of the The authors thank the individuals who participated in this study, family 1 individuals, in whom variable NCVs that blurred as well as E.M. Ross of the University of Texas for kindly providing the division between demyelinating and axonal CMT were a human PLCb2 expression clone, pCMV5-PLCb2. This work was documented. supported by research grants from the National Science Council, GNB4 > In the family carrying the c.158G A (p.Gly53Asp) Taiwan, ROC (NSC97-2314-B-075-064-MY3, NSC98-2320-B-010- mutation, men tended to be more severely affected than 029-MY3, NSC99-2314-B-010-013-MY3, and NSC100-2314- women. If this proves to be true in a larger cohort, it will B-075-020-MY2), the Taiwan Ministry of Education Aim for imply that the Gb4 signal-transduction pathway might the Top University Program (V100-E6-006, V101E7-005, and be influenced by gender-preferential genes in the periph- V102E9-006), the Brain Research Center, National Yang-Ming eral nerve tissues. One recent study uncovered 159 genes University, and the High-throughput Genome Analysis Core that show male or female preference when expressed in Facility and Bioinformatics Consortium of Taiwan of the National the brain.32 Our study suggests the interesting possibility Core Facility Program for Biotechnology, Taiwan (NSC100-2319-B- 010-001 and NSC100-2319-B-010-002). of identifying genes with sex-preferential expression in peripheral nerves. Received: September 6, 2012 The fact that no significant difference in protein stability Revised: November 26, 2012 exists between wild-type and altered forms of Gb4 suggests Accepted: January 19, 2013 GNB4 that the pathomechanism of the mutations is not Published: February 21, 2013 due to a haploinsufficiency of the Gb4 molecules. This finding is consistent with the possible dominant-negative Web Resources mechanism. Because the stability of Gb4 is not affected by the GNB4 mutations, the reduced Gb4 expression in the The URLs for data presented herein are as follows: sural nerves of the individuals with GNB4 mutation 1000 Genomes Project, http://www.1000genomes.org b b suggests that the altered G 4 might be toxic. If the G 4- dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/ related GPCR signaling function is vital to some cells, the GATK, http://www.broadinstitute.org/gatk/ dominant-negative effect of the altered Gb4 could be lethal GRCh37 patch 2, ftp://ftp.ensembl.org/pub/release-61/ or toxic to them. Alternatively, the altered Gb4 might have NHLBI Exome Variant Server Exome Sequencing Project (ESP), gained a toxic function that is unrelated to the dominant- http://evs.gs.washington.edu/EVS/

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