Datasets:

cnachteg

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duvel

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c, d) Sequence chromatograms indicating the wild-type, homozygous affected and heterozygous carrier forms of c) the C to T transition at position c.229 changing the arginine residue to cysteine at position 77 of the @GENE$ protein (c.229C>T; @VARIANT$) and d) the c.238-241delATTG (@VARIANT$) in S100A13. Mutation name is based on the full-length S100A3 (NM_002960) and @GENE$ (NM_001024210) transcripts.

S100A3;2223

S100A13;7523

p.R77C;tmVar:p|SUB|R|77|C;HGVS:p.R77C;VariantGroup:3;CorrespondingGene:6274;RS#:138355706;CA#:1116284

p.I80Gfs*13;tmVar:p|FS|I|80|G|13;HGVS:p.I80GfsX13;VariantGroup:7;CorrespondingGene:6284

0no label

We report digenic variants in SCRIB and PTK7 associated with NTDs in addition to SCRIB and @GENE$ heterozygous variants in additional NTD cases. The combinatorial variation of @GENE$ c.1925C > G (@VARIANT$) and SCRIB c.3323G > A (@VARIANT$) only occurred in one spina bifida case, and was not found in the 1000G database or parental samples of NTD cases.

CELSR1;7665

PTK7;43672

p.P642R;tmVar:p|SUB|P|642|R;HGVS:p.P642R;VariantGroup:5;CorrespondingGene:5754;RS#:148120569;CA#:3816292

p.G1108E;tmVar:p|SUB|G|1108|E;HGVS:p.G1108E;VariantGroup:3;CorrespondingGene:23513;RS#:529610993;CA#:4918763

0no label

The @GENE$-@VARIANT$ variant was previously reported to be associated with LQTS; KCNH2-p.C108Y is a novel variant; and @GENE$-p.K897T and KCNE1-@VARIANT$ were reported to influence the electrical activity of cardiac cells and to act as modifiers of the KCNH2 and KCNQ1 channels.

KCNQ1;85014

KCNH2;201

p.R583H;tmVar:p|SUB|R|583|H;HGVS:p.R583H;VariantGroup:4;CorrespondingGene:3784;RS#:199473482;CA#:6304

p.G38S;tmVar:p|SUB|G|38|S;HGVS:p.G38S;VariantGroup:1;CorrespondingGene:3753;RS#:1805127;CA#:131330

0no label

Among the variants identified in @GENE$, four are known variants, and one, is a novel missense variant at the exon 9 (c.@VARIANT$ p.K953E) present in heterozygosis (Figure 1B). Within the three variants in the coding sequence of @GENE$, two missense variants, both present in heterozygosis, rs3135932 (c.@VARIANT$ p. S159G) and rs2229113 (c.1051 G > A p.G351R), have already been described in the literature.

NOD2;11156

IL10RA;1196

2857A > G;tmVar:c|SUB|A|2857|G;HGVS:c.2857A>G;VariantGroup:0;CorrespondingGene:64127;RS#:8178561;CA#:10006322

475A > G;tmVar:c|SUB|A|475|G;HGVS:c.475A>G;VariantGroup:0;CorrespondingGene:3587;RS#:8178561;CA#:10006322

0no label

Protein structure analysis We performed protein structure analysis on the two @GENE$ mutations (@VARIANT$ and p.G213S) and two novel @GENE$ mutations (@VARIANT$ and p.I312M) that were identified in this study.

WNT10A;22525

EDA;1896

p.R171C;tmVar:p|SUB|R|171|C;HGVS:p.R171C;VariantGroup:3;CorrespondingGene:80326;RS#:116998555;CA#:2113955

p.G257R;tmVar:p|SUB|G|257|R;HGVS:p.G257R;VariantGroup:0;CorrespondingGene:1896;RS#:1057517882;CA#:16043329

0no label

Additionally, a monoallelic C to T transition at nucleotide 511 (@VARIANT$) of the coding sequence in exon 3 of @GENE$ was detected, this leads to the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome showed that the mutant EDA allele was from his mother (Fig. 2C), however, we were unable to screen for WNT10A mutations because of insufficient DNA. "S2" is a 17-year-old boy who had curly hair, 17 missing permanent teeth and hypohidrosis, his skin and nails were normal (Fig. 1 and Table 1). The p.Arg153Cys (c.457C>T) mutation was found in exon 3 of @GENE$, it results in the substitution of @VARIANT$. Moreover, a heterozygous p.Gly213Ser (c.637G>A) mutation was detected in exon 3 of WNT10A, this leads to the substitution of Gly at residue 213 to Ser.

WNT10A;22525

EDA;1896

c.511C>T;tmVar:c|SUB|C|511|T;HGVS:c.511C>T;VariantGroup:3;CorrespondingGene:80326;RS#:116998555;CA#:2113955

Arg at residue 153 to Cys;tmVar:p|SUB|R|153|C;HGVS:p.R153C;VariantGroup:6;CorrespondingGene:1896;RS#:397516662(Expired)

0no label

CSS161458 had a heterozygous splicing variant @VARIANT$ in @GENE$, as described above, and a heterozygous missense variant c.464G>T(@VARIANT$) in MYOD1 was also identified. Although no direct interaction between RIPPLY1 and MYOD1 has been reported, they may together dysregulate the TBX6 pathway given the deleterious nature of both variants (Table 2). DISCUSSION In this study, we performed exome sequencing on 584 patients with @GENE$ and without a molecular diagnosis.

RIPPLY1;138181

CS;56073

c.156-1G>C;tmVar:c|SUB|G|156-1|C;HGVS:c.156-1G>C;VariantGroup:12;CorrespondingGene:92129

p.Arg155Leu;tmVar:p|SUB|R|155|L;HGVS:p.R155L;VariantGroup:2;CorrespondingGene:4654;RS#:757176822;CA#:5906444

0no label

Detection of mutations in @GENE$ and @GENE$ in group I are relatively common in East Asian populations, including Koreans, indicating that application of panel sequencing covering the genes prioritized based on the ethnicity-specific prevalence would be effective for identifying GJB2 single heterozygotes with severe to profound SNHL in Koreans. For the family SH60 with a most likely genetic etiology but without a clear result after TES, whole exome sequencing can be used for definitive molecular diagnosis. This family SH60 segregates prelingual or perilingual severe to profound SNHL, likely in an autosomal dominant fashion, although prelingual SNHL of SH60-136 was caused by autosomal recessive mutations in other deafness genes (Figure 3). Further segregation analyses of the two variants (p.R143W and p.D771N) among the six family members of SH60 as well as clinical evaluations including audiograms excluded both @VARIANT$ of GJB2 and @VARIANT$ of WFS1 as a molecular etiology of SH60-136.

MYO15A;56504

TMC1;23670

p.R143W;tmVar:p|SUB|R|143|W;HGVS:p.R143W;VariantGroup:1;CorrespondingGene:2706;RS#:80338948;CA#:172234

p.D771N;tmVar:p|SUB|D|771|N;HGVS:p.D771N;VariantGroup:13;CorrespondingGene:7466;RS#:534067035;CA#:2839681

0no label

Representative western blot and bar graph showing expression levels of SEC23A (A) and @GENE$ (B) proteins in wild-type (Wt); SEC23A M400I/+ heterozygous; SEC23AM400I/+ MAN1B1R334C/+ double heterozygous; and SEC23A@VARIANT$/M400I MAN1B1R334C/@VARIANT$ double homozygous mutant fibroblasts. The error bars represent standard error of the mean (SEM). Differences in protein levels were detected by one-way ANOVA (analysis of variance), followed by Tukey's multiple comparison test. @GENE$ was used as an internal control. ***, P < 0.001.

MAN1B1;5230

GAPDH;107053

M400I;tmVar:p|SUB|M|400|I;HGVS:p.M400I;VariantGroup:0;CorrespondingGene:10484;RS#:866845715;CA#:259543384

R334C;tmVar:p|SUB|R|334|C;HGVS:p.R334C;VariantGroup:4;CorrespondingGene:11253;RS#:387906886;CA#:129197

0no label

Three rare missense variants (R2034Q, @VARIANT$, and E2003D) of the SPG11 gene were found. The high detection rate of missense variants of this gene is probably due to the large size of the coding region; therefore, we suggest that these SPG11 variants are unlikely to be deleterious. Variants in the @GENE$ gene are most commonly associated with autosomal recessive spastic paraplegia, although homozygous variants have been recently identified in juvenile ALS, and heterozygous missense variants in sALS. Variants in UBQLN2 have been shown to be a cause of dominant X-linked ALS. A previously reported (@VARIANT$,) and a novel variant (Q84H) were found in the UBQLN2 gene. The novel Q84H variant affects the N-terminal ubiquitin-like domain of the @GENE$ protein, which is involved in binding to proteasome subunits.

SPG11;41614

ubiquilin-2;81830

L2118V;tmVar:p|SUB|L|2118|V;HGVS:p.L2118V;VariantGroup:13;CorrespondingGene:80208;RS#:766851227;CA#:7534152

M392V;tmVar:p|SUB|M|392|V;HGVS:p.M392V;VariantGroup:17;CorrespondingGene:29978;RS#:104893941

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (c.757G>A; p.Ala253Thr of @GENE$ and c.488_490delGTT; p.Cys163del of KAL1) and NELF/@GENE$ (@VARIANT$ of NELF and c.824G>A; @VARIANT$ of TACR3).

NELF;10648

TACR3;824

c. 1160-13C>T;tmVar:c|SUB|C|1160-13|T;HGVS:c.1160-13C>T;VariantGroup:5;CorrespondingGene:26012;RS#:781275840;CA#:5370137

p.Trp275X;tmVar:p|SUB|W|275|X;HGVS:p.W275X;VariantGroup:1;CorrespondingGene:6870;RS#:144292455;CA#:144871

0no label

@GENE$ and @GENE$ have similar neurodevelopmental functions and are essential for self-avoidance in the developing mouse retina. In patient AVM144, the compound heterozygous variants @VARIANT$ and c.1000T>A (@VARIANT$) were identified in PTPN13 (table 2).

DSCAML1;79549

DSCAM;74393

c.116-1G>A;tmVar:c|SUB|G|116-1|A;HGVS:c.116-1G>A;VariantGroup:5;CorrespondingGene:83394;RS#:1212415588

p.Ser334Thr;tmVar:p|SUB|S|334|T;HGVS:p.S334T;VariantGroup:0;CorrespondingGene:5783;RS#:755467869;CA#:2995566

0no label

(c, d) @GENE$ @VARIANT$ showed no significant influence on the RNA structure, and the MFE value of SCN5A p.R1865H mutation was approximately similar to that of the wild type Physical and chemical parameter prediction of protein Compared with the amino acids of wild-type KCNH2 (Table 3), @GENE$ @VARIANT$ showed a decreasing trend in molecular weight and increasing instability.

SCN5A;22738

KCNH2;201

p.R1865H;tmVar:p|SUB|R|1865|H;HGVS:p.R1865H;VariantGroup:1;CorrespondingGene:6331;RS#:370694515;CA#:64651

p.307_308del;tmVar:p|DEL|307_308|;HGVS:p.307_308del;VariantGroup:16;CorrespondingGene:3757

0no label

In patient AVM028, one novel heterozygous VUS (@VARIANT$ [p.His736Arg]) in @GENE$ inherited from the father and one likely pathogenic de novo novel heterozygous variant (c.311T>C [@VARIANT$]) in TIMP3 were identified (online supplementary table S2). While @GENE$ blocks VEGF/VEGFR2 signalling, RASA1 modulates differentiation and proliferation of blood vessel endothelial cells downstream of VEGF (figure 3).

RASA1;2168

TIMP3;36322

c.2207A>G;tmVar:c|SUB|A|2207|G;HGVS:c.2207A>G;VariantGroup:6;CorrespondingGene:5921;RS#:1403332745

p.Leu104Pro;tmVar:p|SUB|L|104|P;HGVS:p.L104P;VariantGroup:7;CorrespondingGene:23592;RS#:1290872293

0no label

Interestingly, four of these TEK mutations (@VARIANT$, p.I148T, p.Q214P, and p.G743A) co-occurred with three heterozygous mutations in another major PCG gene CYP1B1 (p.A115P, @VARIANT$, and p.R368H) in five families. The parents of these probands harbored either of the heterozygous TEK or @GENE$ alleles and were asymptomatic, indicating a potential digenic mode of inheritance. Furthermore, we ascertained the interactions of TEK and CYP1B1 by co-transfection and pull-down assays in HEK293 cells. Ligand responsiveness of the wild-type and mutant TEK proteins was assessed in HUVECs using immunofluorescence analysis. We observed that recombinant TEK and CYP1B1 proteins interact with each other, while the disease-associated allelic combinations of TEK (p.E103D)::CYP1B1 (p.A115P), TEK (p.Q214P)::CYP1B1 (p.E229K), and TEK (p.I148T)::CYP1B1 (p.R368H) exhibit perturbed interaction. The mutations also diminished the ability of @GENE$ to respond to ligand stimulation, indicating perturbed TEK signaling.

CYP1B1;68035

TEK;397

p.E103D;tmVar:p|SUB|E|103|D;HGVS:p.E103D;VariantGroup:2;CorrespondingGene:7010;RS#:572527340;CA#:5015873

p.E229K;tmVar:p|SUB|E|229|K;HGVS:p.E229K;VariantGroup:8;CorrespondingGene:1545;RS#:57865060;CA#:145183

0no label

Mutagenesis Sequence variants KCNH2-@VARIANT$ (p.C108Y) and KCNQ1-c.G1748A (@VARIANT$) were introduced into @GENE$ and @GENE$ cDNAs, respectively, as described previously.

KCNH2;201

KCNQ1;85014

c.G323A;tmVar:c|SUB|G|323|A;HGVS:c.323G>A;VariantGroup:3;CorrespondingGene:3757

p.R583H;tmVar:p|SUB|R|583|H;HGVS:p.R583H;VariantGroup:4;CorrespondingGene:3784;RS#:199473482;CA#:6304

11

However, proband P05 also carried a paternal variant (DCC @VARIANT$) and a maternal variant (CCDC88C p. Arg1299Cys). Considering the facts that the loss-of-function mutations in FGFR1 were identified to act in concert with other gene defects and the @GENE$ p. Arg1299Cys variant was reported in a PSIS patient with an IHH-causative gene in a digenic manner, the possibility of oligogenic inheritance in family 05 cannot be ruled out. Six families harbored only one variant of IHH genes, but none had sufficient evidence to be identified as monogenic models. Among these variants, one was frameshift variant, immunoglobulin superfamily member 10 (IGSF10) @VARIANT$, and the rest were missense variants. However, the possibility of being loss-of-function intolerant (pLI) value of @GENE$ is zero, which means that single heterozygous LoF variant of IGSF10 is not sufficient to cause disease.

CCDC88C;18903

IGSF10;18712

p. Gln91Arg;tmVar:p|SUB|Q|91|R;HGVS:p.Q91R;VariantGroup:1;CorrespondingGene:80067;RS#:766366919

p. Thr584Serfs*5;tmVar:p|FS|T|584|S|5;HGVS:p.T584SfsX5;VariantGroup:2;CorrespondingGene:285313;RS#:751845547;CA#:2670482

0no label

Interestingly, they found no disease-related or sudden death in this family, whereas disease-related deaths and sudden deaths were much more common in families carrying the @GENE$ @VARIANT$ variant. The p.E924K variant may, therefore, produce a more subtle phenotype. Morner et al. reported a case in a Swedish patient digenic for MYH7 p.E924K and @GENE$ @VARIANT$ mutations, the carriers of the MYBPC3 variant apparently having late-onset HCM.

MYH7;68044

MYBPC3;215

Arg403Gln;tmVar:p|SUB|R|403|Q;HGVS:p.R403Q;VariantGroup:3;CorrespondingGene:4625;RS#:121913624;CA#:10365

Val896Met;tmVar:p|SUB|V|896|M;HGVS:p.V896M;VariantGroup:7;CorrespondingGene:4607;RS#:35078470;CA#:12837

0no label

Amino acid conservation analysis showed that seven of the 10 variants (CELSR1 p.G1122S, CELSR1 @VARIANT$, @GENE$ @VARIANT$, @GENE$ p.P642R, SCRIB p.G1108E, SCRIB p.G644V and SCRIB p.K618R) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

DVL3;20928

PTK7;43672

p.R769W;tmVar:p|SUB|R|769|W;HGVS:p.R769W;VariantGroup:4;CorrespondingGene:9620;RS#:201601181

p.R148Q;tmVar:p|SUB|R|148|Q;HGVS:p.R148Q;VariantGroup:8;CorrespondingGene:1857;RS#:764021343;CA#:2727085

0no label

It was shown that digenic variants in @GENE$ and @GENE$ contribute to PCG and that variants in both FOXC1 and PITX2 are responsible for some cases of ARS. This prompted us to explore the frequency of CHD in patients with ARS carrying a Foxc1 mutation and whether or not there is a need to carry on WES to investigate the role of other variants in conjunction with FOXC1 that would explain these cardiac defects. Whole Exome Sequencing A tool to draw genotype-phenotype correlation out of the 67 FOXC1 variants reported so far to be linked to the ARS, only nine have been shown to be linked to cardiac defects in addition to the ocular defects. A scrutinized review of the literature of these nine variants, namely p.Q70Hfs*8, p.P79T, @VARIANT$, p. A85P, p.L86F, p.F112S, @VARIANT$, p.G149D, and p.R170W, did show that the cardiac phenotype with which they are associated is not as clear as it is presumed.

CYP1B1;68035

MYOC;220

p.S82T;tmVar:p|SUB|S|82|T;HGVS:p.S82T;VariantGroup:111;CorrespondingGene:6012

p.R127L;tmVar:p|SUB|R|127|L;HGVS:p.R127L;VariantGroup:19;CorrespondingGene:2296;RS#:1085307884

0no label

His cells produced higher levels of IgG and IgM than his mother (II.2, who bears both the TNFRSF13B/TACI @VARIANT$ and TCF3 @VARIANT$ mutations). The combination of @GENE$ T168fsX191and @GENE$/TACI C104R mutations in the proband resulted in a greater net effect that the sum of each individual mutation would predict than the sum of deficits observed for each mutation alone (that is, Ig levelIII.2-(IgIII.2-IgIII.1)+(IgIII.2-IgII.3)).

TCF3;2408

TNFRSF13B;49320

C104R;tmVar:p|SUB|C|104|R;HGVS:p.C104R;VariantGroup:2;CorrespondingGene:23495;RS#:34557412;CA#:117387

T168fsX191;tmVar:p|FS|T|168||191;HGVS:p.T168fsX191;VariantGroup:1;CorrespondingGene:6929

0no label

Apart from carrying TG mutation(s), 6 cases also had mutation(s) in genes associated with DH (SLC26A4, DUOX2, @GENE$ and @GENE$). A total of 6 TPO variants were separately found in 6 patients (6/43, 14%) in heterozygous status. All but 1 patient had a TPO mutation in association with mutation(s) in different genes. A total of 2 novel variants, @VARIANT$ and @VARIANT$, were located in a myeloperoxidase-like domain, the catalytic site of the enzyme (Fig. S3B).

DUOXA2;57037

TPO;461

p.S309P;tmVar:p|SUB|S|309|P;HGVS:p.S309P;VariantGroup:13;CorrespondingGene:2304;RS#:1162674885

p.S571R;tmVar:p|SUB|S|571|R;HGVS:p.S571R;VariantGroup:26;CorrespondingGene:79048;RS#:765990605

0no label

While signal corresponding to myc-pendrin was observed in ~65% of cells, ratio of V5-@GENE$ A372V, L445W, @VARIANT$, or G672E positive cells was significantly decreased (Supplementary Fig. 5a, b). Under these conditions, co-expression of EphA2 did not affect protein expression levels of these pathogenic forms of pendrin (Fig. 5a) but partially restored membrane localization of myc-pendrin A372V, L445W or Q446R (Supplementary Fig. 5a, b). On the other hand, @GENE$ overexpression did not affect localization of G672E. The substitutions of Leu117 to Phe (@VARIANT$), Ser166 to Asn (S166N), and Phe335 to Leu (F335L), identified in Pendred syndrome patients, do not affect their membrane localization.

pendrin;20132

EphA2;20929

Q446R;tmVar:p|SUB|Q|446|R;HGVS:p.Q446R;VariantGroup:15;CorrespondingGene:5172;RS#:768471577;CA#:4432777

L117F;tmVar:p|SUB|L|117|F;HGVS:p.L117F;VariantGroup:18;CorrespondingGene:23985

0no label

We have screened 108 GJB2 heterozygous Chinese patients for mutations in @GENE$ by sequencing. We have excluded the possibility that mutations in exon 1 of @GENE$ and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and @VARIANT$) occurring in compound heterozygosity with the @VARIANT$ and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T).

GJB3;7338

GJB2;2975

A194T;tmVar:c|SUB|A|194|T;HGVS:c.194A>T;VariantGroup:4;CorrespondingGene:2707;RS#:117385606;CA#:118313

235delC;tmVar:c|DEL|235|C;HGVS:c.235delC;VariantGroup:1;CorrespondingGene:2706;RS#:80338943

0no label

We have screened 108 GJB2 heterozygous Chinese patients for mutations in @GENE$ by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of @GENE$ are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (@VARIANT$ and A194T) occurring in compound heterozygosity with the 235delC and @VARIANT$ of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T).

GJB3;7338

GJB6;4936

N166S;tmVar:p|SUB|N|166|S;HGVS:p.N166S;VariantGroup:0;CorrespondingGene:2707;RS#:121908851;CA#:118311

299delAT;tmVar:c|DEL|299|AT;HGVS:c.299delAT;VariantGroup:12;CorrespondingGene:2706

0no label

We identified a novel compound heterozygous variant in BBS1 c.1285dup (p.(Arg429Profs*72); a likely pathogenic novel variant affecting the conserved residue 354 in the functional domain of @GENE$ (@VARIANT$; p.(Asn354Lys)); a pathogenic new homozygous nucleotide change in @GENE$ that leads to a stop codon in position 255, @VARIANT$, and a likely pathogenic homozygous substitution c.1235G > T in BBS6, leading to the change p.(Cys412Phe).

BBS2;12122

BBS7;12395

c.1062C > G;tmVar:c|SUB|C|1062|G;HGVS:c.1062C>G;VariantGroup:22;CorrespondingGene:583

c.763A > T;tmVar:c|SUB|A|763|T;HGVS:c.763A>T;VariantGroup:29;CorrespondingGene:55212

0no label

Variants in all known WS candidate genes (EDN3, EDNRB, @GENE$, @GENE$, SOX10, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (@VARIANT$; p.Arg203Cys) and TYRO3 (c.1037T>A; @VARIANT$) gene was identified in the exome data of both patients.

MITF;4892

PAX3;22494

c.607C>T;tmVar:c|SUB|C|607|T;HGVS:c.607C>T;VariantGroup:1;CorrespondingGene:333929;RS#:149676512;CA#:8229366

p.Ile346Asn;tmVar:p|SUB|I|346|N;HGVS:p.I346N;VariantGroup:2;CorrespondingGene:7301;RS#:12148316;CA#:7494886

0no label

Interestingly, four of these TEK mutations (p.E103D, p.I148T, @VARIANT$, and p.G743A) co-occurred with three heterozygous mutations in another major PCG gene CYP1B1 (@VARIANT$, p.E229K, and p.R368H) in five families. The parents of these probands harbored either of the heterozygous @GENE$ or @GENE$ alleles and were asymptomatic, indicating a potential digenic mode of inheritance.

TEK;397

CYP1B1;68035

p.Q214P;tmVar:p|SUB|Q|214|P;HGVS:p.Q214P;VariantGroup:10;CorrespondingGene:7010

p.A115P;tmVar:p|SUB|A|115|P;HGVS:p.A115P;VariantGroup:0;CorrespondingGene:1545;RS#:764338357;CA#:1620052

11

These two individuals were heterozygous carriers of @VARIANT$ mutation in ABCC6 and @VARIANT$ in GGCX. Since heterozygous carriers of p.R1141X in @GENE$ alone do not manifest PXE and @GENE$ mutations with respect to coagulation disorder are recessive, these findings suggest that the skin phenotype in these two individuals may be due to digenic inheritance.

ABCC6;55559

GGCX;639

p.R1141X;tmVar:p|SUB|R|1141|X;HGVS:p.R1141X;VariantGroup:6;CorrespondingGene:368;RS#:72653706;CA#:129115

p.V255M;tmVar:p|SUB|V|255|M;HGVS:p.V255M;VariantGroup:1;CorrespondingGene:2677;RS#:121909683;CA#:214957

11

Patient P0418 carries a nonsense mutation in USH2A (@VARIANT$) and a missense mutation in @GENE$ (@VARIANT$), but his brother, who is also clinically affected, does not carry the MYO7A mutation. Patient P0432 has a c.4030_4037delATGGCTGG (p.M1344fsX42) mutation in USH2A and a missense mutation in @GENE$ (p.R1189W), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23.

MYO7A;219

CDH23;11142

p.S5030X;tmVar:p|SUB|S|5030|X;HGVS:p.S5030X;VariantGroup:47;CorrespondingGene:7399;RS#:758660532;CA#:1392795

p.K268R;tmVar:p|SUB|K|268|R;HGVS:p.K268R;VariantGroup:135;CorrespondingGene:4647;RS#:184866544;CA#:182406

0no label

Sequence alterations were detected in the COL6A3 (rs144651558), RYR1 (@VARIANT$), CAPN3 (rs138172448), and DES (@VARIANT$) genes. These variants were then screened in his sister who had inherited all variants except that found in the @GENE$ gene. The @GENE$ and RYR1 variants were predicted to be benign by SIFT and PolyPhen and MutationTaster analysis.

CAPN3;52

COL6A3;37917

rs143445685;tmVar:rs143445685;VariantGroup:1;CorrespondingGene:6261;RS#:143445685

rs144901249;tmVar:rs144901249;VariantGroup:3;CorrespondingGene:1674;RS#:144901249

0no label

Hence, @GENE$ mutations can lead to a multisystem proteinopathy although with incomplete penetrance. A single SQSTM1 mutation (c.1165+1G>A) has been linked to MRV in one family and an unrelated patient. This patient was subsequently found to carry a coexisting TIA1 variant (c.1070A>G, @VARIANT$) by Evila et al.. Evila et al.'s study reported also an additional sporadic MRV case carrying the same @GENE$ variant but a different SQSTM1 mutation (@VARIANT$), which is known to cause PDB, ALS, and FTD, but the patient's phenotype was not illustrated.

SQSTM1;31202

TIA1;20692

p.Asn357Ser;tmVar:p|SUB|N|357|S;HGVS:p.N357S;VariantGroup:5;CorrespondingGene:7072;RS#:116621885;CA#:1697407

p.Pro392Leu;tmVar:p|SUB|P|392|L;HGVS:p.P392L;VariantGroup:1;CorrespondingGene:8878;RS#:104893941;CA#:203866

0no label

The large genomic rearrangement in @GENE$ previously reported by Le Guedard et al. was not detected in this group of patients. Three pathogenic or presumably pathogenic mutations in @GENE$ were found in three patients, specifically, an already reported nonsense mutation (p.W38X), a novel nucleotide duplication (c.84dupC; @VARIANT$), and a novel sequence variant (c.46C>G; @VARIANT$).

PCDH15;23401

USH1G;56113

p.D29fsX29;tmVar:p|FS|D|29||29;HGVS:p.D29fsX29;VariantGroup:279;CorrespondingGene:26839

p.L16V;tmVar:p|SUB|L|16|V;HGVS:p.L16V;VariantGroup:18;CorrespondingGene:124590;RS#:876657419;CA#:10576353

0no label

Although our present cohort did not carry homozygous changes in any of the known PCG genes, we reanalyzed our samples that harbored heterozygous mutations in any of these genes along with the @GENE$ mutations. We observed that in 5 PCG cases heterozygous @GENE$ mutations (p.A115P, @VARIANT$, and p.R368H) co-occurred with heterozygous TEK mutations (p.E103D, p.I148T, @VARIANT$, and p.G743A) indicating a potential digenic inheritance (Fig. 1a).

TEK;397

CYP1B1;68035

p.E229 K;tmVar:p|SUB|E|229|K;HGVS:p.E229K;VariantGroup:8;CorrespondingGene:1545;RS#:57865060;CA#:145183

p.Q214P;tmVar:p|SUB|Q|214|P;HGVS:p.Q214P;VariantGroup:10;CorrespondingGene:7010

0no label

Variants in all known WS candidate genes (EDN3, EDNRB, @GENE$, PAX3, SOX10, SNAI2, and @GENE$) were searched and a novel rare heterozygous deletion mutation (c.965delA; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; @VARIANT$) and TYRO3 (c.1037T>A; @VARIANT$) gene was identified in the exome data of both patients.

MITF;4892

TYRO3;4585

p.Arg203Cys;tmVar:p|SUB|R|203|C;HGVS:p.R203C;VariantGroup:1;CorrespondingGene:333929;RS#:149676512;CA#:8229366

p.Ile346Asn;tmVar:p|SUB|I|346|N;HGVS:p.I346N;VariantGroup:2;CorrespondingGene:7301;RS#:12148316;CA#:7494886

0no label

The p.Ile312Met (@VARIANT$) mutation in @GENE$ and heterozygous p.Arg171Cys (c.511C>T) mutation in WNT10A were detected. The coding sequence in exon 9 of EDA showed a C to G transition, which results in the substitution of @VARIANT$; also, the coding sequence in exon 3 of @GENE$ showed a C to T transition at nucleotide 511, which results in the substitution of Arg at residue 171 to Cys.

EDA;1896

WNT10A;22525

c.936C>G;tmVar:c|SUB|C|936|G;HGVS:c.936C>G;VariantGroup:1;CorrespondingGene:80326

Ile at residue 312 to Met;tmVar:p|SUB|I|312|M;HGVS:p.I312M;VariantGroup:7;CorrespondingGene:1896

0no label

RESULTS Mutations at the gap junction proteins @GENE$ and @GENE$ can interact to cause non-syndromic deafness In total, 108 probands screened for mutations in the Cx26 gene were found to carry a single recessive mutant allele. In those samples, no mutation was detected on the second allele either in Cx26-exon-1/splice sites or in GJB6. To investigate the role of GJB3 variations along with GJB2 mutations for a possible combinatory allelic disease inheritance, we have screened patients with heterozygous GJB2 mutations for variants in Cx31 by sequencing. Analysis of the entire coding region of the Cx31 gene revealed the presence of two different missense mutations (N166S and A194T) occurring in compound heterozygosity along with the 235delC and 299delAT of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and @VARIANT$/A194T). In family A, a profoundly hearing impaired proband was found to be heterozygous for a novel A to G transition at nucleotide position 497 of GJB3, resulting in an @VARIANT$ (N166S) and for the 235delC of GJB2 (Fig. 1b, d).

Cx26;2975

Cx31;7338

299delAT;tmVar:c|DEL|299|AT;HGVS:c.299delAT;VariantGroup:12;CorrespondingGene:2706

asparagine into serine substitution in codon 166;tmVar:p|SUB|N|166|S;HGVS:p.N166S;VariantGroup:0;CorrespondingGene:2707;RS#:121908851;CA#:118311

0no label

Sequence analyses of EDA and @GENE$ genes. (A) The @GENE$ mutation @VARIANT$ and WNT10A mutation @VARIANT$ were found in patient N1, who inherited the mutant allele from his mother.

WNT10A;22525

EDA;1896

c.769G>C;tmVar:c|SUB|G|769|C;HGVS:c.769G>C;VariantGroup:0;CorrespondingGene:1896;RS#:1057517882;CA#:16043329

c.511C>T;tmVar:c|SUB|C|511|T;HGVS:c.511C>T;VariantGroup:3;CorrespondingGene:80326;RS#:116998555;CA#:2113955

0no label

These results suggest an important role of ephrin-B2 as an inducer of EphA2 endocytosis with the transmembrane binding partner, pendrin, while its effect is weaker than that of @GENE$. Aberrant regulation of pathogenic forms of @GENE$ via EphA2 Some pathogenic variants of pendrin are not affected by EphA2/ephrin-B2 regulation. a, b Immunoprecipitation of EphA2 with mutated pendrin. myc-pendrin A372V, L445W, Q446R, G672E were not co-immunoprecipitated with EphA2. Densitometric quantifications are shown (b). Mean +- SEM; one-way ANOVA with Bonferroni post hoc analyses; *p < 0.05; (n = 3). c, d Immunoprecipitation of EphA2 with mutated pendrin. Immunocomplex of myc-pendrin L117F, S166N and @VARIANT$ was not affected. Densitometric quantifications are shown (d). Mean +- SEM; (n = 3). e, f Internalization of EphA2 and mutated pendrin triggered by ephrin-B2 stimulation. Pendrin @VARIANT$ was not internalized after ephrin-B2 stimulation while EphA2 and other mutated pendrins were not affected.

ephrin-A1;3262

pendrin;20132

F355L;tmVar:p|SUB|F|355|L;HGVS:p.F355L;VariantGroup:4;CorrespondingGene:1969;RS#:370923409

S166N;tmVar:p|SUB|S|166|N;HGVS:p.S166N;VariantGroup:22;CorrespondingGene:23985

0no label

The p.Ile312Met (c.936C>G) mutation in EDA and heterozygous @VARIANT$ (c.511C>T) mutation in WNT10A were detected. The coding sequence in exon 9 of EDA showed a C to G transition, which results in the substitution of @VARIANT$; also, the coding sequence in exon 3 of WNT10A showed a C to T transition at nucleotide 511, which results in the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome revealed that the mutant alleles were from his mother, who carried digenic heterozygous @GENE$ and @GENE$ mutations at the same locus as that of N2 (Fig. 2B).

EDA;1896

WNT10A;22525

p.Arg171Cys;tmVar:p|SUB|R|171|C;HGVS:p.R171C;VariantGroup:3;CorrespondingGene:80326;RS#:116998555;CA#:2113955

Ile at residue 312 to Met;tmVar:p|SUB|I|312|M;HGVS:p.I312M;VariantGroup:7;CorrespondingGene:1896

11

One heterozygous NELF splice mutation (@VARIANT$) has been described. However, the only KS individual within the family also had a heterozygous @GENE$ mutation (@VARIANT$), suggesting digenic disease. This @GENE$ deletion was associated with exon 10 skipping, but was not sufficient to cause KS alone.

FGFR1;69065

NELF;10648

c.1159-14_22del;tmVar:c|DEL|1159-14_22|;HGVS:c.1159-14_22del;VariantGroup:12;CorrespondingGene:26012

p.Leu342Ser;tmVar:p|SUB|L|342|S;HGVS:p.L342S;VariantGroup:2;CorrespondingGene:2260;RS#:121909638;CA#:130218

0no label

Under these conditions, co-expression of EphA2 did not affect protein expression levels of these pathogenic forms of pendrin (Fig. 5a) but partially restored membrane localization of myc-pendrin @VARIANT$, L445W or Q446R (Supplementary Fig. 5a, b). On the other hand, EphA2 overexpression did not affect localization of G672E. The substitutions of @VARIANT$ (L117F), Ser166 to Asn (S166N), and Phe335 to Leu (F335L), identified in Pendred syndrome patients, do not affect their membrane localization. Given the reported normal function of pendrin L117F and pendrin S166N as an anion exchanger, compromised regulatory machinery of pendrin function may cause the observed symptoms. To examine whether @GENE$ is involved in dysfunction of pendrin caused by these amino acid substitutions, the effect of pendrin L117F, @GENE$ S166N, and pendrin F355L mutations on EphA2 interaction and internalization was examined.

EphA2;20929

pendrin;20132

A372V;tmVar:p|SUB|A|372|V;HGVS:p.A372V;VariantGroup:11;CorrespondingGene:5172;RS#:121908364;CA#:253306

Leu117 to Phe;tmVar:p|SUB|L|117|F;HGVS:p.L117F;VariantGroup:18;CorrespondingGene:23985

0no label

Circles, female; squares, male; gray, @GENE$/TACI C104R mutation; blue TCF3 T168fsX191 mutation (as indicated). The proband (arrow, II.2) is heterozygous for both the @GENE$ T168fsX191 and TNFRSF13B/TACI C104R mutations. Other family members who have inherited TCF3 T168fsX191 and TNFRSF13B/TACI @VARIANT$ mutations are shown. CVID, common variable immunodeficiency disorder; SLE, systemic lupus erythematosus; sIgAD, selective IgA deficiency; T1D, Type 1 Diabetes, sHGUS, symptomatic hypogammglobulinaemia of uncertain significance; WT, wild-type. (b) Electropherograms showing the T168fsX191 mutation of TCF3 and C104R (c.310T>C) mutation of TACI gene in the proband II.2. The proband's son (III.1) has inherited the TCF3 @VARIANT$ mutation, but not the TNFRSF13B/TACI C104R mutation.

TNFRSF13B;49320

TCF3;2408

C104R;tmVar:p|SUB|C|104|R;HGVS:p.C104R;VariantGroup:2;CorrespondingGene:23495;RS#:34557412;CA#:117387

T168fsX191;tmVar:p|FS|T|168||191;HGVS:p.T168fsX191;VariantGroup:1;CorrespondingGene:6929

0no label

Since TTC26 is an intraflagellar transport (IFT) protein in cilia, we aimed to identify potential interactions between @GENE$ and TTC26. Using coimmunoprecipitation assays, we found that the myc-tagged mutant p.R50C and @VARIANT$ @GENE$ proteins pulled down the Flag-tagged mutant p.A2282T and @VARIANT$ FLNB proteins, respectively (figure 2D, E).

FLNB;37480

TTC26;11786

p.R197C;tmVar:p|SUB|R|197|C;HGVS:p.R197C;VariantGroup:32;CorrespondingGene:79989

p.R566L;tmVar:p|SUB|R|566|L;HGVS:p.R566L;VariantGroup:1;CorrespondingGene:2317;RS#:778577280

0no label

The @VARIANT$ nonsense variant was first detected in compound heterozygous form in a family with two affected siblings suffering from infantile ascending spastic paralysis with bulbar involvement. The ages of onset of the patients with the ALS2 variants reported in this study were later than juvenile ALS onset, which generally manifests before 25 years of age. Previous studies suggested that heterozygous variants in the @GENE$ may be causative for adult-onset sALS. @GENE$ encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, S275N) were detected. The @VARIANT$ variant affects the b isoform of the MATR3 protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms.

ALS2;23264

MATR3;7830

G1177X;tmVar:p|SUB|G|1177|X;HGVS:p.G1177X;VariantGroup:0;CorrespondingGene:57679;RS#:386134180;CA#:356568

P11S;tmVar:p|SUB|P|11|S;HGVS:p.P11S;VariantGroup:6;RS#:995345187

0no label

Sequence analyses of EDA and @GENE$ genes. (A) The EDA mutation c.769G>C and WNT10A mutation c.511C>T were found in patient N1, who inherited the mutant allele from his mother. (B) The EDA mutation @VARIANT$ and WNT10A mutation c.511C>T were found in patient N2, who also inherited the mutant allele from his mother. (C) The @GENE$ mutation c.252DelT and WNT10A mutation c.511C>T were found in patient S1, who inherited the mutant EDA allele from his mother; WNT10A mutations in the parents could not be analyzed. (D) The EDA mutation @VARIANT$ and WNT10A mutation c.637G>A were found in patient S2, who also inherited the mutant allele from his mother; however, his father's DNA sample could not be obtained for analysis.

WNT10A;22525

EDA;1896

c.936C>G;tmVar:c|SUB|C|936|G;HGVS:c.936C>G;VariantGroup:1;CorrespondingGene:80326

c.457C>T;tmVar:c|SUB|C|457|T;HGVS:c.457C>T;VariantGroup:6;CorrespondingGene:1896;RS#:397516662(Expired)

0no label

Variants in all known WS candidate genes (EDN3, EDNRB, MITF, PAX3, @GENE$, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; @VARIANT$) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; p.Arg203Cys) and @GENE$ (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

SOX10;5055

TYRO3;4585

p.Asn322fs;tmVar:p|FS|N|322||;HGVS:p.N322fsX;VariantGroup:3;CorrespondingGene:4286

c.1037T>A;tmVar:c|SUB|T|1037|A;HGVS:c.1037T>A;VariantGroup:2;CorrespondingGene:7301;RS#:12148316;CA#:7494886

0no label

Deleterious variants in HS1BP3 (NM_022460.3: c.94C>A, @VARIANT$) and GNA14 (NM_004297.3: @VARIANT$, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP. Deleterious variants in DNAH17,@GENE$,CAPN11,VPS13C,UNC13B,SPTBN4,@GENE$, and MRPL15 were found in two or more independent pedigrees.

TRPV4;11003

MYOD1;7857

p.Gly32Cys;tmVar:p|SUB|G|32|C;HGVS:p.G32C;VariantGroup:25;CorrespondingGene:64342

c.989_990del;tmVar:c|DEL|989_990|;HGVS:c.989_990del;VariantGroup:16;CorrespondingGene:9630;RS#:750424668;CA#:5094137

0no label

Interestingly, one FALS proband carried 3 variants, each of which has previously been reported as pathogenic: SOD1 @VARIANT$, @GENE$ @VARIANT$, and DCTN1 p.T1249I. Nine apparently sporadic subjects had variants in multiple genes (Table 4), but only two were well-established ALS mutations: @GENE$ p.G287S was found in combination with VAPB p.M170I while a subject with juvenile-onset ALS carried a de novo FUS p.P525L mutation with a paternally-inherited intermediate-sized CAG expansion in ATXN2.

ANG;74385

TARDBP;7221

p.G38R;tmVar:p|SUB|G|38|R;HGVS:p.G38R;VariantGroup:50;CorrespondingGene:6647;RS#:121912431;CA#:257311

p.P136L;tmVar:p|SUB|P|136|L;HGVS:p.P136L;VariantGroup:7;CorrespondingGene:283;RS#:121909543;CA#:258112

0no label

Variants in all known WS candidate genes (EDN3, EDNRB, @GENE$, @GENE$, SOX10, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; @VARIANT$) and TYRO3 (c.1037T>A; @VARIANT$) gene was identified in the exome data of both patients.

MITF;4892

PAX3;22494

p.Arg203Cys;tmVar:p|SUB|R|203|C;HGVS:p.R203C;VariantGroup:1;CorrespondingGene:333929;RS#:149676512;CA#:8229366

p.Ile346Asn;tmVar:p|SUB|I|346|N;HGVS:p.I346N;VariantGroup:2;CorrespondingGene:7301;RS#:12148316;CA#:7494886

0no label

Moreover, a heterozygous p.Gly213Ser (c.637G>A) mutation was detected in exon 3 of @GENE$, this leads to the substitution of @VARIANT$. Sequence analyses revealed that both mutant alleles were from his mother (Fig. 2D), who had a very mild phenotype of isolated tooth agenesis. His father did not have mutations in either of these genes. "S3" is a 14-year-old girl who had the typical clinical characteristics of HED: sparse hair, 26 missing permanent teeth, hypohidrosis, dry skin, and eczema on her body, but no plantar hyperkeratosis or nail abnormalities (Table 1). The heterozygous p.Arg156Cys (c.466C>T) mutation was found in exon 3 of @GENE$, it results in the substitution of @VARIANT$. Additionally, the monoallelic p.Gly213Ser (c.637G>A) mutation was also detected in exon 3 of WNT10A, it results in the substitution of Gly at residue 213 to Ser.

WNT10A;22525

EDA;1896

Gly at residue 213 to Ser;tmVar:p|SUB|G|213|S;HGVS:p.G213S;VariantGroup:4;CorrespondingGene:80326;RS#:147680216;CA#:211313

Arg at residue 156 to Cys;tmVar:p|SUB|R|156|C;HGVS:p.R156C;VariantGroup:5;CorrespondingGene:1896;RS#:132630313;CA#:255655

0no label

Analysis of the proband's exome revealed four potential disease-causing mutations in FTA candidate genes: three heterozygous missense variants in @GENE$ (g.68531T>G, c.503T>G, @VARIANT$; g.112084C>G, c.2450C>G, p.Ser817Cys; g.146466A>G, c.4333A>G, p.Met1445Val) and one in @GENE$ (g.14712G>A, @VARIANT$, p.Gly213Ser) (Figure 2A and Figure S2A,B).

LRP6;1747

WNT10A;22525

p.Met168Arg;tmVar:p|SUB|M|168|R;HGVS:p.M168R;VariantGroup:9;CorrespondingGene:4040

c.637G>A;tmVar:c|SUB|G|637|A;HGVS:c.637G>A;VariantGroup:7;CorrespondingGene:80326;RS#:147680216;CA#:211313

11

We observed that in 5 PCG cases heterozygous @GENE$ mutations (p.A115P, @VARIANT$, and p.R368H) co-occurred with heterozygous @GENE$ mutations (p.E103D, @VARIANT$, p.Q214P, and p.G743A) indicating a potential digenic inheritance (Fig. 1a).

CYP1B1;68035

TEK;397

p.E229 K;tmVar:p|SUB|E|229|K;HGVS:p.E229K;VariantGroup:8;CorrespondingGene:1545;RS#:57865060;CA#:145183

p.I148T;tmVar:p|SUB|I|148|T;HGVS:p.I148T;VariantGroup:5;CorrespondingGene:7010;RS#:35969327;CA#:5015918

11

p.R1110Q was the most common mutation identified in our patient cohort, which differed from previous reports in Korean (p.G488R) and Japanese (@VARIANT$) populations. Additionally, p.K530X was the most common mutation identified in Chinese patients from southern or central China. Besides DUOX2, TG anomalies are another common cause of DH. However, in the present study, four detected @GENE$ variants presented separately in four different patients with heterozygosity and always cooccurred with variants in DUOX2 or other DH-related genes, indicating that the contributions of TG mutations to DH in Xinjiang Han Chinese might be less important. More CH-associated @GENE$ mutations were found recently. Our study identified two known truncating variants, p.Y246X and @VARIANT$, which cooccurred in a patient with permanent CH.

TG;2430

DUOXA2;57037

p.R855Q;tmVar:p|SUB|R|855|Q;HGVS:p.R855Q;VariantGroup:45;CorrespondingGene:50506

p.Y138X;tmVar:p|SUB|Y|138|X;HGVS:p.Y138X;VariantGroup:14;CorrespondingGene:405753;RS#:778410503;CA#:7539391

0no label

In family A, a profoundly hearing impaired proband was found to be heterozygous for a novel A to G transition at nucleotide position 497 of @GENE$, resulting in an asparagine into serine substitution in codon 166 (N166S) and for the 235delC of @GENE$ (Fig. 1b, d). Genotyping analysis revealed that the GJB2/@VARIANT$ was inherited from the unaffected father and the @VARIANT$ of GJB3 was inherited from the normal hearing mother (Fig. 1a).

GJB3;7338

GJB2;2975

235delC;tmVar:c|DEL|235|C;HGVS:c.235delC;VariantGroup:1;CorrespondingGene:2706;RS#:80338943

N166S;tmVar:p|SUB|N|166|S;HGVS:p.N166S;VariantGroup:0;CorrespondingGene:2707;RS#:121908851;CA#:118311

0no label

GFP-CYP1B1 R368H also exhibited relatively reduced ability to immunoprecipitate HA-TEK @VARIANT$ (~70%). No significant change was observed with HA-@GENE$ G743A with GFP-@GENE$ @VARIANT$ as compared to WT proteins (Fig. 2).

TEK;397

CYP1B1;68035

I148T;tmVar:p|SUB|I|148|T;HGVS:p.I148T;VariantGroup:5;CorrespondingGene:7010;RS#:35969327;CA#:5015918

E229 K;tmVar:p|SUB|E|229|K;HGVS:p.E229K;VariantGroup:8;CorrespondingGene:1545;RS#:57865060;CA#:145183

0no label

A rare variant in @GENE$, c.428C>T; @VARIANT$, was detected in Case 2 and was classified as VUS. Finally, for Case 7 and her father, a previously reported @GENE$/FOG2 (c.1632G>A; @VARIANT$) pathogenic missense alteration was identified.

AMH;68060

ZFPM2;8008

p.Thr143Ile;tmVar:p|SUB|T|143|I;HGVS:p.T143I;VariantGroup:3;CorrespondingGene:268;RS#:139265145;CA#:9062862

p.Met544Ile;tmVar:p|SUB|M|544|I;HGVS:p.M544I;VariantGroup:1;CorrespondingGene:23414;RS#:187043152;CA#:170935

0no label

Variants in all known WS candidate genes (EDN3, EDNRB, MITF, PAX3, @GENE$, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; @VARIANT$) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; @VARIANT$) and @GENE$ (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients.

SOX10;5055

TYRO3;4585

p.Asn322fs;tmVar:p|FS|N|322||;HGVS:p.N322fsX;VariantGroup:3;CorrespondingGene:4286

p.Arg203Cys;tmVar:p|SUB|R|203|C;HGVS:p.R203C;VariantGroup:1;CorrespondingGene:333929;RS#:149676512;CA#:8229366

0no label

Interestingly, one FALS proband carried 3 variants, each of which has previously been reported as pathogenic: SOD1 @VARIANT$, ANG p.P136L, and DCTN1 @VARIANT$. Nine apparently sporadic subjects had variants in multiple genes (Table 4), but only two were well-established ALS mutations: @GENE$ p.G287S was found in combination with @GENE$ p.M170I while a subject with juvenile-onset ALS carried a de novo FUS p.P525L mutation with a paternally-inherited intermediate-sized CAG expansion in ATXN2.

TARDBP;7221

VAPB;36163

p.G38R;tmVar:p|SUB|G|38|R;HGVS:p.G38R;VariantGroup:50;CorrespondingGene:6647;RS#:121912431;CA#:257311

p.T1249I;tmVar:p|SUB|T|1249|I;HGVS:p.T1249I;VariantGroup:53;CorrespondingGene:1639;RS#:72466496;CA#:119583

0no label

In our study, we identified four genetic variants in three genes (@GENE$-@VARIANT$, KCNH2-p.C108Y, KCNH2-p.K897T, and @GENE$-@VARIANT$).

KCNQ1;85014

KCNE1;3753

p.R583H;tmVar:p|SUB|R|583|H;HGVS:p.R583H;VariantGroup:4;CorrespondingGene:3784;RS#:199473482;CA#:6304

p.G38S;tmVar:p|SUB|G|38|S;HGVS:p.G38S;VariantGroup:1;CorrespondingGene:3753;RS#:1805127;CA#:131330

11

The mutations of @GENE$ @VARIANT$ and @GENE$ @VARIANT$ were found in the proband by WES and validated as positive by Sanger sequencing.

KCNH2;201

SCN5A;22738

p.307_308del;tmVar:p|DEL|307_308|;HGVS:p.307_308del;VariantGroup:16;CorrespondingGene:3757

p.R1865H;tmVar:p|SUB|R|1865|H;HGVS:p.R1865H;VariantGroup:1;CorrespondingGene:6331;RS#:370694515;CA#:64651

11

Variants in all known WS candidate genes (EDN3, @GENE$, MITF, PAX3, SOX10, SNAI2, and @GENE$) were searched and a novel rare heterozygous deletion mutation (c.965delA; @VARIANT$) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; @VARIANT$) and TYRO3 (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients.

EDNRB;89

TYRO3;4585

p.Asn322fs;tmVar:p|FS|N|322||;HGVS:p.N322fsX;VariantGroup:3;CorrespondingGene:4286

p.Arg203Cys;tmVar:p|SUB|R|203|C;HGVS:p.R203C;VariantGroup:1;CorrespondingGene:333929;RS#:149676512;CA#:8229366

0no label

We identified four genetic variants (@GENE$-p.R583H, KCNH2-p.C108Y, @GENE$-p.K897T, and KCNE1-@VARIANT$) in an LQTS family. On the basis of in silico analysis, clinical data from our family, and the evidence from previous studies, we analyzed two mutated channels, KCNQ1-p.R583H and KCNH2-p.C108Y, using the whole-cell patch clamp technique. We found that KCNQ1-p.R583H was not associated with a severe functional impairment, whereas KCNH2-@VARIANT$, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type.

KCNQ1;85014

KCNH2;201

p.G38S;tmVar:p|SUB|G|38|S;HGVS:p.G38S;VariantGroup:1;CorrespondingGene:3753;RS#:1805127;CA#:131330

p.C108Y;tmVar:p|SUB|C|108|Y;HGVS:p.C108Y;VariantGroup:3;CorrespondingGene:3757

0no label

Two different GJB3 mutations (@VARIANT$ and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (@VARIANT$/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric @GENE$/@GENE$ connexons.

Cx26;2975

Cx31;7338

N166S;tmVar:p|SUB|N|166|S;HGVS:p.N166S;VariantGroup:0;CorrespondingGene:2707;RS#:121908851;CA#:118311

235delC;tmVar:c|DEL|235|C;HGVS:c.235delC;VariantGroup:1;CorrespondingGene:2706;RS#:80338943

0no label

To sum up, SH166-367, SH170-377, and SB175-334 which would have been considered DFNB1 without TES were found to be DFNB7/11, @GENE$, and DFNB16, respectively. Finally, a subject with the heterozygous @VARIANT$ mutation in GJB2 (SH60-136) carried a @VARIANT$ variant in @GENE$ (WFS1) (NM_001145853) according to TES.

DFNB3;56504

Wolfram syndrome 1;4380

p.R143W;tmVar:p|SUB|R|143|W;HGVS:p.R143W;VariantGroup:1;CorrespondingGene:2706;RS#:80338948;CA#:172234

p.D771N;tmVar:p|SUB|D|771|N;HGVS:p.D771N;VariantGroup:13;CorrespondingGene:7466;RS#:534067035;CA#:2839681

0no label

The nucleotide sequence showed a T deletion at nucleotide 252 (c.252DelT) of the coding sequence in exon 1 of @GENE$; this leads to a frame shift from residue 84 and a premature termination at residue 90. Additionally, a monoallelic C to T transition at nucleotide 511 (c.511C>T) of the coding sequence in exon 3 of @GENE$ was detected, this leads to the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome showed that the mutant EDA allele was from his mother (Fig. 2C), however, we were unable to screen for WNT10A mutations because of insufficient DNA. "S2" is a 17-year-old boy who had curly hair, 17 missing permanent teeth and hypohidrosis, his skin and nails were normal (Fig. 1 and Table 1). The @VARIANT$ (c.457C>T) mutation was found in exon 3 of EDA, it results in the substitution of Arg at residue 153 to Cys. Moreover, a heterozygous @VARIANT$ (c.637G>A) mutation was detected in exon 3 of WNT10A, this leads to the substitution of Gly at residue 213 to Ser.

EDA;1896

WNT10A;22525

p.Arg153Cys;tmVar:p|SUB|R|153|C;HGVS:p.R153C;VariantGroup:6;CorrespondingGene:1896;RS#:397516662(Expired)

p.Gly213Ser;tmVar:p|SUB|G|213|S;HGVS:p.G213S;VariantGroup:4;CorrespondingGene:80326;RS#:147680216;CA#:211313

0no label

In patient AVM144, the compound heterozygous variants c.116-1G>A and c.1000T>A (@VARIANT$) were identified in PTPN13 (table 2). Potential oligogenic inheritance Variants in more than one gene (at least one likely pathogenic variant) with differing inheritance origin were identified in three patients (figure 1). In patient AVM558, a pathogenic heterozygous variant c.920dupA (p.Asn307LysfsTer27) inherited from the mother was identified in ENG. Another de novo novel heterozygous missense variant, @VARIANT$ (p.Arg565Gln), was identified in @GENE$ (online supplementary table S2), which encodes the kinase responsible for phosphorylation of residue T312 in SMAD1 to block its activity in BMP/TGF-beta signalling. This de novo variant may modify the effect of the truncating variant in ENG by repressing @GENE$/TGF-beta signalling.

MAP4K4;7442

BMP;55955

p.Ser334Thr;tmVar:p|SUB|S|334|T;HGVS:p.S334T;VariantGroup:0;CorrespondingGene:5783;RS#:755467869;CA#:2995566

c.1694G>A;tmVar:c|SUB|G|1694|A;HGVS:c.1694G>A;VariantGroup:5;CorrespondingGene:9448;RS#:1212415588

0no label

Case Description: We previously described a Proband and her brother (P1, P2) with unusually severe CH associated with a DUOX2 homozygous nonsense mutation (@VARIANT$); P1, P2: thyrotropin >100 microU/mL [reference range (RR) 0.5 to 6.3]; and P1: free T4 (FT4) <0.09 ng/dL (RR 0.9 to 2.3). Subsequent studies have revealed a homozygous DUOX1 mutation (@VARIANT$) resulting in aberrant splicing and a protein truncation (p.Val607Aspfs*43), which segregates with CH in this kindred. Conclusion: This is a report of digenic mutations in DUOX1 and DUOX2 in association with CH, and we hypothesize that the inability of @GENE$ to compensate for @GENE$ deficiency in this kindred may underlie the severe CH phenotype.

DUOX1;68136

DUOX2;9689

p.R434*;tmVar:p|SUB|R|434|*;HGVS:p.R434*;VariantGroup:0;CorrespondingGene:50506;RS#:119472026

c.1823-1G>C;tmVar:c|SUB|G|1823-1|C;HGVS:c.1823-1G>C;VariantGroup:17;CorrespondingGene:53905

0no label

DISCUSSION We present a Chinese family with PFBC in which the previously reported heterozygous mutation @VARIANT$ (p.His596Arg) in @GENE$ and the SNP (@VARIANT$) c.317G>C (p.Arg106Pro) in PDGFRB were identified. The proband's father with the SLC20A2 c.1787A>G (p.His596Arg) mutation showed obvious brain calcification but was clinically asymptomatic. The proband's mother with the @GENE$ c.317G>C (p.Arg106Pro) variant showed very slight calcification and was clinically asymptomatic.

SLC20A2;68531

PDGFRB;1960

c.1787A>G;tmVar:c|SUB|A|1787|G;HGVS:c.1787A>G;VariantGroup:2;CorrespondingGene:6575

rs544478083;tmVar:rs544478083;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

0no label

Whole genome SNP genotyping, whole exome sequencing and segregation analysis using Sanger approach was performed and a novel single nucleotide deletion mutation (@VARIANT$) in the @GENE$ gene and a rare heterozygous, missense damaging variant (@VARIANT$; p.Val34Gly) in the @GENE$ was identified.

MITF;4892

C2orf74;49849

c.965delA;tmVar:c|DEL|965|A;HGVS:c.965delA;VariantGroup:4;CorrespondingGene:4286

c.101T>G;tmVar:c|SUB|T|101|G;HGVS:c.101T>G;VariantGroup:0;CorrespondingGene:339804;RS#:565619614;CA#:1674263

11

A single SQSTM1 mutation (@VARIANT$) has been linked to MRV in one family and an unrelated patient. This patient was subsequently found to carry a coexisting @GENE$ variant (c.1070A>G, @VARIANT$) by Evila et al.. Evila et al.'s study reported also an additional sporadic MRV case carrying the same TIA1 variant but a different @GENE$ mutation (p.Pro392Leu), which is known to cause PDB, ALS, and FTD, but the patient's phenotype was not illustrated.

TIA1;20692

SQSTM1;31202

c.1165+1G>A;tmVar:c|SUB|G|1165+1|A;HGVS:c.1165+1G>A;VariantGroup:8;CorrespondingGene:8878;RS#:796051870(Expired)

p.Asn357Ser;tmVar:p|SUB|N|357|S;HGVS:p.N357S;VariantGroup:5;CorrespondingGene:7072;RS#:116621885;CA#:1697407

0no label

GFP-CYP1B1 @VARIANT$ also exhibited relatively reduced ability to immunoprecipitate HA-TEK I148T (~70%). No significant change was observed with HA-@GENE$ @VARIANT$ with GFP-@GENE$ E229 K as compared to WT proteins (Fig. 2).

TEK;397

CYP1B1;68035

R368H;tmVar:p|SUB|R|368|H;HGVS:p.R368H;VariantGroup:1;CorrespondingGene:1545;RS#:79204362;CA#:119016

G743A;tmVar:c|SUB|G|743|A;HGVS:c.743G>A;VariantGroup:12;CorrespondingGene:7010;RS#:202131936;CA#:5016449

0no label

We provide evidence that mutations in the @GENE$ and @GENE$ genes can interact to cause hearing loss in digenic heterozygotes. RESULTS Mutations at the gap junction proteins Cx26 and Cx31 can interact to cause non-syndromic deafness In total, 108 probands screened for mutations in the Cx26 gene were found to carry a single recessive mutant allele. In those samples, no mutation was detected on the second allele either in Cx26-exon-1/splice sites or in GJB6. To investigate the role of GJB3 variations along with GJB2 mutations for a possible combinatory allelic disease inheritance, we have screened patients with heterozygous GJB2 mutations for variants in Cx31 by sequencing. Analysis of the entire coding region of the Cx31 gene revealed the presence of two different missense mutations (N166S and A194T) occurring in compound heterozygosity along with the 235delC and 299delAT of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and @VARIANT$/@VARIANT$).

Cx26;2975

Cx31;7338

299delAT;tmVar:c|DEL|299|AT;HGVS:c.299delAT;VariantGroup:12;CorrespondingGene:2706

A194T;tmVar:c|SUB|A|194|T;HGVS:c.194A>T;VariantGroup:4;CorrespondingGene:2707;RS#:117385606;CA#:118313

11

Amino acid conservation analysis showed that seven of the 10 variants (@GENE$ @VARIANT$, CELSR1 p.R769W, DVL3 p.R148Q, @GENE$ @VARIANT$, SCRIB p.G1108E, SCRIB p.G644V and SCRIB p.K618R) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

CELSR1;7665

PTK7;43672

p.G1122S;tmVar:p|SUB|G|1122|S;HGVS:p.G1122S;VariantGroup:0;CorrespondingGene:9620;RS#:200363699;CA#:10295026

p.P642R;tmVar:p|SUB|P|642|R;HGVS:p.P642R;VariantGroup:5;CorrespondingGene:5754;RS#:148120569;CA#:3816292

0no label

Three rare missense variants (R2034Q, L2118V, and @VARIANT$) of the SPG11 gene were found. The high detection rate of missense variants of this gene is probably due to the large size of the coding region; therefore, we suggest that these SPG11 variants are unlikely to be deleterious. Variants in the SPG11 gene are most commonly associated with autosomal recessive spastic paraplegia, although homozygous variants have been recently identified in juvenile ALS, and heterozygous missense variants in sALS. Variants in UBQLN2 have been shown to be a cause of dominant X-linked ALS. A previously reported (M392V,) and a novel variant (@VARIANT$) were found in the @GENE$ gene. The novel Q84H variant affects the N-terminal ubiquitin-like domain of the ubiquilin-2 protein, which is involved in binding to proteasome subunits. @GENE$ variants have been mostly detected in familial ALS cases that are localized within the C-terminus of the FUS protein.

UBQLN2;81830

FUS;2521

E2003D;tmVar:p|SUB|E|2003|D;HGVS:p.E2003D;VariantGroup:3;CorrespondingGene:80208;RS#:954483795

Q84H;tmVar:p|SUB|Q|84|H;HGVS:p.Q84H;VariantGroup:43;CorrespondingGene:29978

0no label

The nucleotide sequence showed a T deletion at nucleotide 252 (c.252DelT) of the coding sequence in exon 1 of EDA; this leads to a frame shift from residue 84 and a premature @VARIANT$. Additionally, a monoallelic C to T transition at nucleotide 511 (@VARIANT$) of the coding sequence in exon 3 of WNT10A was detected, this leads to the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome showed that the mutant EDA allele was from his mother (Fig. 2C), however, we were unable to screen for WNT10A mutations because of insufficient DNA. "S2" is a 17-year-old boy who had curly hair, 17 missing permanent teeth and hypohidrosis, his skin and nails were normal (Fig. 1 and Table 1). The p.Arg153Cys (c.457C>T) mutation was found in exon 3 of @GENE$, it results in the substitution of Arg at residue 153 to Cys. Moreover, a heterozygous p.Gly213Ser (c.637G>A) mutation was detected in exon 3 of @GENE$, this leads to the substitution of Gly at residue 213 to Ser.

EDA;1896

WNT10A;22525

termination at residue 90;tmVar:p|Allele|X|90;VariantGroup:10;CorrespondingGene:1896

c.511C>T;tmVar:c|SUB|C|511|T;HGVS:c.511C>T;VariantGroup:3;CorrespondingGene:80326;RS#:116998555;CA#:2113955

0no label

Sequence alterations were detected in the COL6A3 (rs144651558), RYR1 (@VARIANT$), CAPN3 (rs138172448), and DES (@VARIANT$) genes. These variants were then screened in his sister who had inherited all variants except that found in the CAPN3 gene. The @GENE$ and RYR1 variants were predicted to be benign by SIFT and PolyPhen and MutationTaster analysis. The @GENE$ variant c.1663G>A (rs138172448) results in a p.Val555Ile change.

COL6A3;37917

CAPN3;52

rs143445685;tmVar:rs143445685;VariantGroup:1;CorrespondingGene:6261;RS#:143445685

rs144901249;tmVar:rs144901249;VariantGroup:3;CorrespondingGene:1674;RS#:144901249

0no label

The nucleotide sequence showed a G to C transition at nucleotide 769 (c.769G>C) of the coding sequence in exon 7 of EDA, which results in the substitution of @VARIANT$. Additionally, the nucleotide sequence showed a monoallelic C to T transition at nucleotide 511 (c.511C>T) of the coding sequence in exon 3 of WNT10A, which results in the substitution of @VARIANT$. DNA sequencing of the parents' genome revealed that both mutant alleles were from their mother (Fig. 2A), who carried a heterozygous @GENE$ mutation (c.769G>C) and a heterozygous WNT10A c.511C>T mutation, and showed absence of only the left upper lateral incisor without other clinical abnormalities. No mutations in these genes were found in the father. Sequence analyses of EDA and @GENE$ genes.

EDA;1896

WNT10A;22525

Gly at residue 257 to Arg;tmVar:p|SUB|G|257|R;HGVS:p.G257R;VariantGroup:0;CorrespondingGene:1896;RS#:1057517882;CA#:16043329

Arg at residue 171 to Cys;tmVar:p|SUB|R|171|C;HGVS:p.R171C;VariantGroup:3;CorrespondingGene:80326;RS#:116998555;CA#:2113955

0no label

Moreover, a heterozygous p.Gly213Ser (c.637G>A) mutation was detected in exon 3 of @GENE$, this leads to the substitution of @VARIANT$. Sequence analyses revealed that both mutant alleles were from his mother (Fig. 2D), who had a very mild phenotype of isolated tooth agenesis. His father did not have mutations in either of these genes. "S3" is a 14-year-old girl who had the typical clinical characteristics of HED: sparse hair, 26 missing permanent teeth, hypohidrosis, dry skin, and eczema on her body, but no plantar hyperkeratosis or nail abnormalities (Table 1). The heterozygous @VARIANT$ (c.466C>T) mutation was found in exon 3 of @GENE$, it results in the substitution of Arg at residue 156 to Cys.

WNT10A;22525

EDA;1896

Gly at residue 213 to Ser;tmVar:p|SUB|G|213|S;HGVS:p.G213S;VariantGroup:4;CorrespondingGene:80326;RS#:147680216;CA#:211313

p.Arg156Cys;tmVar:p|SUB|R|156|C;HGVS:p.R156C;VariantGroup:5;CorrespondingGene:1896;RS#:132630313;CA#:255655

0no label

The proband (arrow, II.2) is heterozygous for both the @GENE$ @VARIANT$ and TNFRSF13B/TACI @VARIANT$ mutations. Other family members who have inherited TCF3 T168fsX191 and @GENE$/TACI C104R mutations are shown.

TCF3;2408

TNFRSF13B;49320

T168fsX191;tmVar:p|FS|T|168||191;HGVS:p.T168fsX191;VariantGroup:1;CorrespondingGene:6929

C104R;tmVar:p|SUB|C|104|R;HGVS:p.C104R;VariantGroup:2;CorrespondingGene:23495;RS#:34557412;CA#:117387

0no label

Variants in all known WS candidate genes (EDN3, EDNRB, MITF, PAX3, SOX10, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; @VARIANT$) was identified in the @GENE$ gene in both patients. Moreover, heterozygous missense variants in @GENE$ (c.607C>T; p.Arg203Cys) and TYRO3 (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

MITF;4892

SNAI3;8500

p.Asn322fs;tmVar:p|FS|N|322||;HGVS:p.N322fsX;VariantGroup:3;CorrespondingGene:4286

c.1037T>A;tmVar:c|SUB|T|1037|A;HGVS:c.1037T>A;VariantGroup:2;CorrespondingGene:7301;RS#:12148316;CA#:7494886

0no label

The alsin protein encoded by the @GENE$ gene is involved in endosome/membrane trafficking and fusion, cytoskeletal organization, and neuronal development/maintenance. Both homozygous and compound heterozygous variants in the ALS2 gene have been described as causative for juvenile ALS. The @VARIANT$ nonsense variant was first detected in compound heterozygous form in a family with two affected siblings suffering from infantile ascending spastic paralysis with bulbar involvement. The ages of onset of the patients with the ALS2 variants reported in this study were later than juvenile ALS onset, which generally manifests before 25 years of age. Previous studies suggested that heterozygous variants in the ALS2 may be causative for adult-onset sALS. MATR3 encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, S275N) were detected. The @VARIANT$ variant affects the b isoform of the @GENE$ protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms.

ALS2;23264

MATR3;7830

G1177X;tmVar:p|SUB|G|1177|X;HGVS:p.G1177X;VariantGroup:0;CorrespondingGene:57679;RS#:386134180;CA#:356568

P11S;tmVar:p|SUB|P|11|S;HGVS:p.P11S;VariantGroup:6;RS#:995345187

0no label

Three patients carried missense variants both in @GENE$ and other PCP-associated genes: 01F552 (FZD6 @VARIANT$ and @GENE$ @VARIANT$), 335F07 (FZD6 c.544G>A and 2 FAT4 missense variants c.5792A>G; c.10384A>G), and 465F99 (rare FZD1 missense variant c.211C>T and a novel FAT4 missense variant c.10147G>A).

FZD;8321;8323

CELSR2;1078

c.1531C>T;tmVar:c|SUB|C|1531|T;HGVS:c.1531C>T;VariantGroup:29;CorrespondingGene:8323;RS#:151339003;CA#:129147

c.3800A>G;tmVar:c|SUB|A|3800|G;HGVS:c.3800A>G;VariantGroup:2;CorrespondingGene:1952;RS#:373263457;CA#:4677776

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (@VARIANT$; p.Ala253Thr of NELF and c.488_490delGTT; @VARIANT$ of KAL1) and NELF/TACR3 (c. 1160-13C>T of @GENE$ and c.824G>A; p.Trp275X of @GENE$).

NELF;10648

TACR3;824

c.757G>A;tmVar:c|SUB|G|757|A;HGVS:c.757G>A;VariantGroup:3;CorrespondingGene:26012;RS#:142726563;CA#:5370407

p.Cys163del;tmVar:p|DEL|163|C;HGVS:p.163delC;VariantGroup:10;CorrespondingGene:3730

0no label

These mutations are expected to affect the three classes of @GENE$ isoforms (Tables 2, 3, Figure 1). Eight pathogenic or presumably pathogenic mutations in @GENE$ were found in six patients, specifically, a previously reported mutation that affects splicing (c.6050-9G>A), a novel nucleotide deletion (c.6404_6405delAG; @VARIANT$), and six missense mutations, four of which (p.R1189W, p.R1379P, @VARIANT$, and p.R3043W) had not been previously reported.

harmonin;77476

CDH23;11142

p.E2135fsX31;tmVar:p|FS|E|2135||31;HGVS:p.E2135fsX31;VariantGroup:3;CorrespondingGene:64072;RS#:55947063

p.D2639G;tmVar:p|SUB|D|2639|G;HGVS:p.D2639G;VariantGroup:219;CorrespondingGene:65217

0no label

Subsequently, genetic testing for the LQT1, LQT2, LQT3, LQT5, and @GENE$ genes identified a heterozygous @VARIANT$ (@VARIANT$) mutation of the KCNH2 gene (LQT2) and a heterozygous c.170T > C (p.Ile57Thr) unclassified variant (UV) of the KCNE2 gene (LQT6). The UV (missense mutation) of the KCNE2 gene is likely a pathogenic mutation, what results in the digenic inheritance of @GENE$ and LQT6.

LQT6;71688

LQT2;201

c.3092_3096dup;tmVar:c|DUP|3092_3096||;HGVS:c.3092_3096dup;VariantGroup:2;CorrespondingGene:9992

p.Arg1033ValfsX26;tmVar:p|FS|R|1033|V|26;HGVS:p.R1033VfsX26;VariantGroup:1;CorrespondingGene:3757

0no label

We identified a novel compound heterozygous variant in BBS1 c.1285dup (p.(Arg429Profs*72); a likely pathogenic novel variant affecting the conserved residue 354 in the functional domain of @GENE$ (c.1062C > G; p.(Asn354Lys)); a pathogenic new homozygous nucleotide change in BBS7 that leads to a @VARIANT$, c.763A > T, and a likely pathogenic homozygous substitution c.1235G > T in @GENE$, leading to the change p.(@VARIANT$).

BBS2;12122

BBS6;10318

stop codon in position 255;tmVar:p|Allele|X|255;VariantGroup:1;CorrespondingGene:79738;RS#:139658279

Cys412Phe;tmVar:p|SUB|C|412|F;HGVS:p.C412F;VariantGroup:15;CorrespondingGene:8195;RS#:1396840386

0no label

@VARIANT$ lies within a cytoplasmic domain between the 5th-6th transmembrane domains of this G-protein coupled receptor, thereby predicting the loss of 191AA from codons 275-465 and truncating ~40% of the C-terminus (Figure 1C). He had no mutations in CHD7, @GENE$, FGFR1, PROK2, PROKR2, TAC3, KAL1, GNRHR, @GENE$, or KISS1R. Unfortunately, in all three probands with NELF mutations, no other family members were available for de novo or segregation analysis. Discussion Our findings indicate that NELF is likely to be causative in IHH/KS. Previously, Miura et al demonstrated a heterozygous c.1438A>G (@VARIANT$) NELF variant in 1/65 IHH patients based upon sequence AY_255128 (now revised to c.1432A>G ;p.Thr478Ala from NP_056352).

FGF8;7715

GNRH1;641

Trp275;tmVar:p|Allele|W|275;VariantGroup:1;CorrespondingGene:6870;RS#:144292455

p.Thr480Ala;tmVar:p|SUB|T|480|A;HGVS:p.T480A;VariantGroup:0;CorrespondingGene:26012;RS#:121918340;CA#:130174

0no label

Genotype + and - indicate in the figure wild type and mutated, respectively. (B) Percentage of CD3+CD4+@GENE$+ cells after PMA stimulation (mean +- SEM of n = 2). (C) Sanger sequencing confirmed a homozygous in-frame deletion (@VARIANT$) in @GENE$ gene and a homozygous splice-donor mutation (@VARIANT$) in CARD9 gene.

IL17;1651

MYD88;1849

c.195_197delGGA;tmVar:p|DEL|195_197|G;HGVS:p.195_197delG;VariantGroup:2;CorrespondingGene:4615

c.1434+1G>C;tmVar:c|SUB|G|1434+1|C;HGVS:c.1434+1G>C;VariantGroup:0;CorrespondingGene:64170;RS#:141992399;CA#:500026

0no label

The combinatorial variation of @GENE$ c.1925C > G (@VARIANT$) and SCRIB c.3323G > A (@VARIANT$) only occurred in one spina bifida case, and was not found in the 1000G database or parental samples of NTD cases. Location analysis of missense changes showed that p.G1108E was located very close to the fourth PDZ domain (1109-1192) of @GENE$. The PDZ domains of human SCRIB are required for correct localization and physical interaction with other proteins, such as the core PCP protein VANGL2, which is required for transducing PCP signals.

PTK7;43672

SCRIB;44228

p.P642R;tmVar:p|SUB|P|642|R;HGVS:p.P642R;VariantGroup:5;CorrespondingGene:5754;RS#:148120569;CA#:3816292

p.G1108E;tmVar:p|SUB|G|1108|E;HGVS:p.G1108E;VariantGroup:3;CorrespondingGene:23513;RS#:529610993;CA#:4918763

0no label

Exome analysis for the proband identified three sequence variants in FTA candidate genes, two in LRP6 (g.27546T>A, c.379T>A, p.Ser127Thr; g.124339A>G, c.3224A>G, p.Asn1075Ser) and one in @GENE$ (@VARIANT$, c.499G>C, p.Glu167Gln) (Figure 4A). The @GENE$ @VARIANT$ mutation is a rare variant with an MAF of 0.0024 in EAS.

WNT10A;22525

LRP6;1747

g.14574G>C;tmVar:g|SUB|G|14574|C;HGVS:g.14574G>C;VariantGroup:5;CorrespondingGene:80326;RS#:148714379

c.3224A>G;tmVar:c|SUB|A|3224|G;HGVS:c.3224A>G;VariantGroup:8;CorrespondingGene:4040;RS#:202124188

0no label

All exons including the 5' and 3' untranslated regions of the @GENE$, @GENE$, SCN5A, KCNE1, and KCNE2 genes were amplified by polymerase chain reaction (PCR). The PCR products were screened for mutations by direct sequencing (Applied Biosystems Big-Dye Terminator v1.1 cycle sequencing kit; Life Technologies, Grand Island, NY, USA) with an Applied Biosystems Prism 3100 DNA Analyzer (Life Technologies, Grand Island, NY, USA) using previously reported protocols. In silico predictions of pathogenicity were made using the Alamut Visual software (Interactive Biosoftware, Rouen, France), and population allele frequencies were determined using the ExAC Browser (Beta) database. 4.3. Mutagenesis Sequence variants KCNH2-@VARIANT$ (p.C108Y) and KCNQ1-@VARIANT$ (p.R583H) were introduced into KCNH2 and KCNQ1 cDNAs, respectively, as described previously.

KCNQ1;85014

KCNH2;201

c.G323A;tmVar:c|SUB|G|323|A;HGVS:c.323G>A;VariantGroup:3;CorrespondingGene:3757

c.G1748A;tmVar:c|SUB|G|1748|A;HGVS:c.1748G>A;VariantGroup:4;CorrespondingGene:3784;RS#:199473482;CA#:6304

0no label

Patient P0432 has a c.4030_4037delATGGCTGG (p.M1344fsX42) mutation in USH2A and a missense mutation in CDH23 (@VARIANT$), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in @GENE$ (@VARIANT$), USH1G (c.46C>G; p.L16V) and @GENE$ (c.9921T>G).

MYO7A;219

USH2A;66151

p.R1189W;tmVar:p|SUB|R|1189|W;HGVS:p.R1189W;VariantGroup:61;CorrespondingGene:64072;RS#:745855338;CA#:5544764

c.6657T>C;tmVar:c|SUB|T|6657|C;HGVS:c.6657T>C;VariantGroup:153;CorrespondingGene:4647

0no label

All six affected family members, and none of the three unaffected persons, had a heterozygous @GENE$ @VARIANT$/p.Asn197Ilefs*81 mutation as well as a heterozygous @GENE$ c.1559G>A/@VARIANT$ mutation.

ENAM;9698

LAMA3;18279

c.588 +1delG;tmVar:c|DEL|588+1|G;HGVS:c.588+1delG;VariantGroup:9;CorrespondingGene:13801

p.Cys520Tyr;tmVar:p|SUB|C|520|Y;HGVS:p.C520Y;VariantGroup:6;CorrespondingGene:3909

11

Her fasting C-peptide was 0.86 ng/mL (reference range: 0.5-3 ng/dL) and 60-minute stimulated C-peptide was 1.96 ng/mL. Due to the negative diabetes autoantibody panel, she underwent genetic testing as part of the SEARCH monogenic diabetes ancillary study at 11 years of age demonstrating a heterozygous missense mutation in exon 4 of HNF4A, R127W (@VARIANT$) and a heterozygous frameshift mutation in exon 4 of @GENE$, P291fsinsC (@VARIANT$). @GENE$ therapy was completely discontinued and she was started on glipizide (1.25 mg once daily) with the dose titrated to 2.5 mg once daily based on blood sugar checks, with weekly blood sugar reviews and close support from a diabetes specialist nurse practitioner.

HNF1A;459

Insulin;173

c.379C>T;tmVar:c|SUB|C|379|T;HGVS:c.379C>T;VariantGroup:3;CorrespondingGene:3172;RS#:370239205;CA#:9870226

c.872dup;tmVar:c|DUP|872||;HGVS:c.872dup;VariantGroup:1;CorrespondingGene:6927;RS#:587776825

0no label

Under these conditions, co-expression of @GENE$ did not affect protein expression levels of these pathogenic forms of pendrin (Fig. 5a) but partially restored membrane localization of myc-pendrin A372V, @VARIANT$ or Q446R (Supplementary Fig. 5a, b). On the other hand, EphA2 overexpression did not affect localization of G672E. The substitutions of Leu117 to Phe (L117F), Ser166 to Asn (S166N), and @VARIANT$ (F335L), identified in Pendred syndrome patients, do not affect their membrane localization. Given the reported normal function of pendrin L117F and pendrin S166N as an anion exchanger, compromised regulatory machinery of pendrin function may cause the observed symptoms. To examine whether EphA2 is involved in dysfunction of @GENE$ caused by these amino acid substitutions, the effect of pendrin L117F, pendrin S166N, and pendrin F355L mutations on EphA2 interaction and internalization was examined.

EphA2;20929

pendrin;20132

L445W;tmVar:p|SUB|L|445|W;HGVS:p.L445W;VariantGroup:0;CorrespondingGene:5172;RS#:111033307;CA#:253309

Phe335 to Leu;tmVar:p|SUB|F|335|L;HGVS:p.F335L;VariantGroup:20;CorrespondingGene:13836

0no label

Most intriguingly, the more severely affected of the two patients, Patient 3, was found to harbor a known heterozygous variant in @GENE$ (@VARIANT$, p.Met703Leu, rs121908603:A>C), a testes determining gene associated with heart anomalies, and this variant has been previously reported in an individual with diaphragmatic hernia (Bleyl et al., 2007). We postulate that the cumulative effect of these changes in two different genes may be contributing to the patient's more severe phenotype. In a second example, we identified a monoallelic change in SRD5A2 (c.G680A, p.Arg227Gln, rs9332964:G>A), in conjunction with the @VARIANT$ of @GENE$. Monoallelic inheritance of SRD5A2, although uncommon, has been reported in a severely under-virilized individual with hypospadias and bilateral inguinal testes (Chavez, Ramos, Gomez, & Vilchis, 2014).

ZFPM2;8008

SF1;138518

c.A2107C;tmVar:c|SUB|A|2107|C;HGVS:c.2107A>C;VariantGroup:3;CorrespondingGene:23414;RS#:121908603;CA#:117963

single amino acid deletion at position 372;tmVar:|Allele|SINGLEAMINO|372;VariantGroup:20;CorrespondingGene:7536

0no label

Variants in all known WS candidate genes (EDN3, @GENE$, MITF, PAX3, SOX10, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (@VARIANT$; p.Arg203Cys) and TYRO3 (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients. Variant in SNAI3 (c.607C>T; p.Arg203Cys) gene is rare in population and is probably damaging and deleterious as predicted by PolyPhen2 and SIFT, respectively. Variant in @GENE$ (@VARIANT$; p.Ile346Asn) gene is present in population databases with high frequency (0.22 MAF) and is benign and tolerated as predicted by PolyPhen2 and SIFT, respectively.

EDNRB;89

TYRO3;4585

c.607C>T;tmVar:c|SUB|C|607|T;HGVS:c.607C>T;VariantGroup:1;CorrespondingGene:333929;RS#:149676512;CA#:8229366

c.1037T>A;tmVar:c|SUB|T|1037|A;HGVS:c.1037T>A;VariantGroup:2;CorrespondingGene:7301;RS#:12148316;CA#:7494886

0no label

In patient AVM558, a pathogenic heterozygous variant @VARIANT$ (p.Asn307LysfsTer27) inherited from the mother was identified in @GENE$. Another de novo novel heterozygous missense variant, @VARIANT$ (p.Arg565Gln), was identified in MAP4K4 (online supplementary table S2), which encodes the kinase responsible for phosphorylation of residue T312 in @GENE$ to block its activity in BMP/TGF-beta signalling.

ENG;92

SMAD1;21196

c.920dupA;tmVar:c|DUP|920|A|;HGVS:c.920dupA;VariantGroup:12;CorrespondingGene:2022

c.1694G>A;tmVar:c|SUB|G|1694|A;HGVS:c.1694G>A;VariantGroup:5;CorrespondingGene:9448;RS#:1212415588

0no label

The nucleotide sequence showed a @VARIANT$ (c.252DelT) of the coding sequence in exon 1 of EDA; this leads to a frame shift from residue 84 and a premature termination at residue 90. Additionally, a monoallelic C to T transition at nucleotide 511 (c.511C>T) of the coding sequence in exon 3 of @GENE$ was detected, this leads to the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome showed that the mutant @GENE$ allele was from his mother (Fig. 2C), however, we were unable to screen for WNT10A mutations because of insufficient DNA. "S2" is a 17-year-old boy who had curly hair, 17 missing permanent teeth and hypohidrosis, his skin and nails were normal (Fig. 1 and Table 1). The p.Arg153Cys (c.457C>T) mutation was found in exon 3 of EDA, it results in the substitution of Arg at residue 153 to Cys. Moreover, a heterozygous @VARIANT$ (c.637G>A) mutation was detected in exon 3 of WNT10A, this leads to the substitution of Gly at residue 213 to Ser.

WNT10A;22525

EDA;1896

T deletion at nucleotide 252;tmVar:c|Allele|T|252;VariantGroup:9;CorrespondingGene:1896

p.Gly213Ser;tmVar:p|SUB|G|213|S;HGVS:p.G213S;VariantGroup:4;CorrespondingGene:80326;RS#:147680216;CA#:211313

0no label