Datasets:

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A comprehensive epilepsy gene panel, including deletion/duplication analysis, revealed variants of unknown significance in @GENE$ (NM_00156.4, c.79T>C, @VARIANT$), @GENE$ (NM_018328.4, @VARIANT$, p.Leu667Trp), and NRXN1 (NM_004801.4, c.2686C>T, p.Arg896Trp), all of which were inherited.

GAMT;32089

MBD5;81861

p.Tyr27His;tmVar:p|SUB|Y|27|H;HGVS:p.Y27H;VariantGroup:0;CorrespondingGene:2593;RS#:200833152;CA#:295620

c.2000T>G;tmVar:c|SUB|T|2000|G;HGVS:c.2000T>G;VariantGroup:3;CorrespondingGene:55777;RS#:796052711;CA#:315814

0no label

Interestingly, one FALS proband carried 3 variants, each of which has previously been reported as pathogenic: SOD1 p.G38R, @GENE$ @VARIANT$, and @GENE$ @VARIANT$. Nine apparently sporadic subjects had variants in multiple genes (Table 4), but only two were well-established ALS mutations: TARDBP 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

DCTN1;3011

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

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

11

None of 2,504 self-declared healthy individuals in TGP has both @GENE$, c.1070A > G (@VARIANT$) and @GENE$, c.1175C > T (@VARIANT$).

TIA1;20692

SQSTM1;31202

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

11

21 Additional gene reportedly linked to tumorigenesis include RYR3, 22 @GENE$, 23 @GENE$, 24 and CAPN9. 25 The RYR3 (NM_001036: c.7812C > G, p.Asn2604Lys) and EBNA1BP2 (NM_001159936: c.1034A > T, @VARIANT$) variants were classified as likely benign and benign, respectively, while the TRIP6 (NM_003302: c.822G > C, @VARIANT$) and the CAPN9 (NM_006615: c.55G > T, p.Ala19Ser) variants were classified as VUS.

EBNA1BP2;4969

TRIP6;37757

p.Asn345Ile;tmVar:p|SUB|N|345|I;HGVS:p.N345I;VariantGroup:5;CorrespondingGene:10969;RS#:11559312;CA#:803919

p.Glu274Asp;tmVar:p|SUB|E|274|D;HGVS:p.E274D;VariantGroup:22;CorrespondingGene:7205;RS#:76826261;CA#:4394675

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 (@VARIANT$; p.Arg203Cys) and TYRO3 (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

MITF;4892

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 addition, 2 genes presented variants in 3 patients: @GENE$ (patients 6, 7 and 8) and @GENE$ (patients 1, 4 and 8). Furthermore, RIPK4 presented 2 variants in patient 1. Finally, BNC2 variant @VARIANT$:p.(Pro623His) (MAF = 0.002) was detected in 2 patients (patient 1 and 7) and MAML3 variant c.881A>G:@VARIANT$ (MAF = 0.0028) in patients 7 and 8 ( Table 2 ).

MAML3;41284

NOTCH1;32049

c.1868C>A;tmVar:c|SUB|C|1868|A;HGVS:c.1868C>A;VariantGroup:11;CorrespondingGene:54796;RS#:114596065;CA#:204322

p.(Asn294Ser);tmVar:p|SUB|N|294|S;HGVS:p.N294S;VariantGroup:16;CorrespondingGene:55534;RS#:115966590;CA#:3085269

0no label

We identified a novel variant in the NOD2 gene (c.2857A > G @VARIANT$) and two already described missense variants in the @GENE$ gene (S159G and @VARIANT$). The new @GENE$ missense variant was examined in silico with two online bioinformatics tools to predict the potentially deleterious effects of the mutation.

IL10RA;1196

NOD2;11156

p.K953E;tmVar:p|SUB|K|953|E;HGVS:p.K953E;VariantGroup:0;CorrespondingGene:64127;RS#:8178561

G351R;tmVar:p|SUB|G|351|R;HGVS:p.G351R;VariantGroup:0;CorrespondingGene:3587;RS#:8178561

0no label

We observed that in 5 PCG cases heterozygous @GENE$ mutations (@VARIANT$, p.E229 K, and p.R368H) co-occurred with heterozygous TEK mutations (p.E103D, p.I148T, p.Q214P, and p.G743A) indicating a potential digenic inheritance (Fig. 1a). None of the normal controls carried both the heterozygous combinations of CYP1B1 and TEK mutations. The @GENE$ Q214P and @VARIANT$ alleles were absent in 1024 controls, whereas very low frequencies of heterozygous TEK E103D (0.005) and I148T (0.016) alleles were found in the control population (Table 1).

CYP1B1;68035

TEK;397

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

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

0no label

In the individual carrying the @VARIANT$ NEFH variant, an additional novel alteration (C335R) was detected in the GRN gene. Loss-of-function GRN variants are primarily considered to cause frontotemporal lobar degeneration, but there is evidence that missense GRN variants are also linked to the pathogenesis of ALS. The novel @GENE$ variant reported in this study results in a cysteine-to-arginine change in the cysteine-rich granulin A domain. Four cases were identified to carry @GENE$ variants: the P392L in two cases and the @VARIANT$ and R393Q in single patients.

GRN;1577

SQSTM1;31202

P505L;tmVar:p|SUB|P|505|L;HGVS:p.P505L;VariantGroup:22;CorrespondingGene:4744;RS#:1414968372

E389Q;tmVar:p|SUB|E|389|Q;HGVS:p.E389Q;VariantGroup:24;CorrespondingGene:8878;RS#:1391182750

0no label

Five anencephaly cases carried rare or novel CELSR1 missense variants, three of whom carried additional rare potentially damaging PCP variants: 01F377 (CELSR1 c.6362G>A and PRICKLE4 @VARIANT$), 2F07 (CELSR1 c.8807C>T and DVL3 c.1622C>T), 618F05 (@GENE$ c.8282C>T and SCRIB c.3979G>A). One patient (f93-80) had a novel PTK7 missense variant (c.655A>G) with a rare CELSR2 missense variant (c.1892C>T). Three patients carried missense variants both in @GENE$ and other PCP-associated genes: 01F552 (FZD6 c.1531C>T and CELSR2 c.3800A>G), 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 @VARIANT$).

CELSR1;7665

FZD;8321;8323

c.730C>G;tmVar:c|SUB|C|730|G;HGVS:c.730C>G;VariantGroup:12;CorrespondingGene:29964;RS#:141478229;CA#:3802865

c.10147G>A;tmVar:c|SUB|G|10147|A;HGVS:c.10147G>A;VariantGroup:11;CorrespondingGene:2068;RS#:543855329

0no label

The genotypes of @GENE$ (NM_001257180.2: c.1787A>G, @VARIANT$) and @GENE$ (NM_002609.4: @VARIANT$, p.Arg106Pro) for available individuals are shown.

SLC20A2;68531

PDGFRB;1960

p.His596Arg;tmVar:p|SUB|H|596|R;HGVS:p.H596R;VariantGroup:2;CorrespondingGene:6575

c.317G>C;tmVar:c|SUB|G|317|C;HGVS:c.317G>C;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

11

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

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

0no label

Quantification of cells with dilated endoplasmic reticulum (ER) and COPII vesicles associated with Golgi by transmission electron microscopy Genotype of cell line Cells with dilated ER (%) Cells with Golgi-associated vesicles (%) Wt (N = 414) 2 (0.5) 309 (75) SEC23Ac.1200G>C/+ (N = 83) 83 (100***) 9 (11***) SEC23Ac.1200G>C/+ MAN1B1@VARIANT$/+ (N = 190) 190 (100***) 3 (1.6***) SEC23Ac.1200G>C/c.@VARIANT$; MAN1B1c.1000C>T/c.1000C>T (N = 328) 328 (100***) 2 (0.6***) Increased Intracellular and Secreted Pro-COL1A1 in Fibroblasts with Homozygous Mutations in Both @GENE$ and @GENE$ in the Presence of l-Ascorbic Acid SEC23A is required for normal transport of pro-COL1A1, a major extracellular matrix component of bone.

SEC23A;4642

MAN1B1;5230

c.1000C>T;tmVar:c|SUB|C|1000|T;HGVS:c.1000C>T;VariantGroup:4;CorrespondingGene:11253;RS#:387906886;CA#:129197

1200G>C;tmVar:c|SUB|G|1200|C;HGVS:c.1200G>C;VariantGroup:0;CorrespondingGene:10484;RS#:866845715;CA#:259543384

0no label

Two affected (II-3 and III-9) individuals were selected for WES. +/+, wild-type; +/-, heterozygous for @GENE$ c.109C>T. (b) Electropherograms of unaffected family member (II-2) and subject with @GENE$ (II-3). (c) Multiple sequence alignment shows evolutionary conservation of @VARIANT$ among vertebrates TOR2A missense variant A TOR2A nonsynonymous SNV (@VARIANT$ [NM_130459.3], p.Arg190Cys [NP_569726.2]) was identified in three subjects with BSP and three asymptomatic members from a four generation pedigree (Figure 5; Tables 1, 5, 8 and S2; Data S1).

REEP4;11888

BSP+;3644

Arg37;tmVar:p|Allele|R|37;VariantGroup:10;CorrespondingGene:80346;RS#:780399718

c.568C>T;tmVar:c|SUB|C|568|T;HGVS:c.568C>T;VariantGroup:12;CorrespondingGene:27433;RS#:376074923;CA#:5250615

0no label

Three rare missense variants (R2034Q, @VARIANT$, and E2003D) of the @GENE$ 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 @GENE$ 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 UBQLN2 gene.

SPG11;41614

UBQLN2;81830

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

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

0no label

However, when combined with the @GENE$ mutations, it led to a severe phenotype of thirteen missing teeth in the proband. This genetic synergism is also supported by the potential digenic inheritance of LRP6 and @GENE$ mutations in Family 4. The proband, who had LRP6 p.(@VARIANT$), p.(Ser127Thr), and WNT10A p.(@VARIANT$) variants, showed ten missing teeth, while her parents, who passed individual mutant alleles, had no missing teeth but microdontia and dysmorphology of specific teeth.

LRP6;1747

WNT10A;22525

Asn1075Ser;tmVar:p|SUB|N|1075|S;HGVS:p.N1075S;VariantGroup:8;CorrespondingGene:4040;RS#:202124188

Glu167Gln;tmVar:p|SUB|E|167|Q;HGVS:p.E167Q;VariantGroup:5;CorrespondingGene:80326;RS#:148714379

0no label

The detected @VARIANT$ variant affects the nuclear localization signal 2 (amino acids 568-574) of the @GENE$ protein. A previously characterized pathogenic nonsense variant (G1177X) and a rare missense alteration (@VARIANT$) were detected in the ALS2 gene, both in heterozygous form. The @GENE$ protein encoded by the ALS2 gene is involved in endosome/membrane trafficking and fusion, cytoskeletal organization, and neuronal development/maintenance.

CCNF;1335

alsin;23264

R572W;tmVar:p|SUB|R|572|W;HGVS:p.R572W;VariantGroup:25;CorrespondingGene:899;RS#:199743115;CA#:7842683

R1499H;tmVar:p|SUB|R|1499|H;HGVS:p.R1499H;VariantGroup:4;CorrespondingGene:57679;RS#:566436589;CA#:2057559

0no label

To examine whether EphA2 is involved in dysfunction of pendrin caused by these amino acid substitutions, the effect of pendrin L117F, pendrin S166N, and pendrin F355L mutations on @GENE$ interaction and internalization was examined. While the amount of co-precipitated pendrin mutants with EphA2 was comparable to that of wild type (wt) pendrin (Fig. 5c, d), the S166N mutant failed to be internalized after @GENE$ stimulation (Fig. 5e, f). Taken together, these results further demonstrate that EphA2 could control both pendrin recruitment to the plasma membrane and pendrin exclusion from the plasma membrane. EPHA2 mutations in pendred syndrome patients Identification and characterization of EphA2 mutation from hearing loss patients with EVA. a, b Pedigree chart of the patients carrying mono-allelic EPHA2 and SLC26A4 mutations. c Audiograms of the patient with mono-allelic EPHA2 @VARIANT$ and SLC26A4 p.T410M mutations. d Temporal bone computed tomography (CT) scan of the patient with mono-allelic EPHA2 p.T511M and SLC26A4 @VARIANT$ mutations.

EphA2;20929

ephrin-B2;3019

p.T511M;tmVar:p|SUB|T|511|M;HGVS:p.T511M;VariantGroup:5;CorrespondingGene:1969;RS#:55747232;CA#:625151

p.T410M;tmVar:p|SUB|T|410|M;HGVS:p.T410M;VariantGroup:9;CorrespondingGene:5172;RS#:111033220;CA#:261403

0no label

The nucleotide sequence showed a G to C transition at nucleotide 769 (@VARIANT$) of the coding sequence in exon 7 of EDA, which results in the substitution of Gly at residue 257 to Arg. Additionally, the nucleotide sequence showed a monoallelic C to T transition at nucleotide 511 (c.511C>T) of the coding sequence in exon 3 of WNT10A, which results in the substitution of @VARIANT$. DNA sequencing of the parents' genome revealed that both mutant alleles were from their mother (Fig. 2A), who carried a heterozygous EDA mutation (c.769G>C) and a heterozygous @GENE$ 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 @GENE$ and WNT10A genes.

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

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

To the best of our knowledge, two of the identified variants (@GENE$: c.1183C>A, @VARIANT$; and @GENE$: c.535C>A, @VARIANT$) have not been previously identified.

FOXC2;21091

PITX2;55454

p.(H395N);tmVar:p|SUB|H|395|N;HGVS:p.H395N;VariantGroup:8;CorrespondingGene:2303

p.(P179T);tmVar:p|SUB|P|179|T;HGVS:p.P179T;VariantGroup:3;CorrespondingGene:1545;RS#:771076928

0no label

The nucleotide sequence showed a @VARIANT$ (c.769G>C) of the coding sequence in exon 7 of @GENE$, which results in the substitution of Gly at residue 257 to Arg. Additionally, the nucleotide sequence showed a monoallelic C to T transition at nucleotide 511 (@VARIANT$) of the coding sequence in exon 3 of WNT10A, which results in the substitution of Arg at residue 171 to Cys. DNA sequencing of the parents' genome revealed that both mutant alleles were from their mother (Fig. 2A), who carried a heterozygous EDA mutation (c.769G>C) and a heterozygous @GENE$ c.511C>T mutation, and showed absence of only the left upper lateral incisor without other clinical abnormalities.

EDA;1896

WNT10A;22525

G to C transition at nucleotide 769;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

Compared to WT (wild-type) proteins, we found that the ability of GFP-CYP1B1 A115P and GFP-CYP1B1 E229K to immunoprecipitate HA-@GENE$ E103D and HA-TEK @VARIANT$, respectively, was significantly diminished. GFP-CYP1B1 R368H also exhibited relatively reduced ability to immunoprecipitate HA-TEK I148T (~70%). No significant change was observed with HA-TEK G743A with GFP-CYP1B1 @VARIANT$ as compared to WT proteins (Fig. 2). The WT and mutant TEK proteins expressed at similar levels in the cells, indicating that the mutations did not affect the expression or stability of the proteins (Fig. 2). We also tested the potential of the mutant TEK and @GENE$ proteins to associate with wild-type CYP1B1 and TEK, respectively.

TEK;397

CYP1B1;68035

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

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

0no label

Sequence alterations were detected in the COL6A3 (rs144651558), RYR1 (rs143445685), @GENE$ (@VARIANT$), and @GENE$ (@VARIANT$) genes.

CAPN3;52

DES;56469

rs138172448;tmVar:rs138172448;VariantGroup:2;CorrespondingGene:825;RS#:138172448

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

0no label

Causative heterozygous mutations in @GENE$ (p.N382S/@VARIANT$) and @GENE$ (p.I1176L/@VARIANT$) were identified by whole exome sequencing performed on III-1 and IV-1.

GFI1;3854

MYO6;56417

c.1145A > G;tmVar:c|SUB|A|1145|G;HGVS:c.1145A>G;VariantGroup:1;CorrespondingGene:2672;RS#:28936381;CA#:119872

c.3526A > C;tmVar:c|SUB|A|3526|C;HGVS:c.3526A>C;VariantGroup:2;CorrespondingGene:4646;RS#:755922465;CA#:141060203

11

In the present study, we found two variants: the E758K variant in two patients and the @VARIANT$ variant in one case, with both variants located within the coiled-coil domain (amino acid positions 331-906) of the protein, which is not in line with previous findings. Without additional functional evidence, the pathogenicity of these variants is uncertain. Three rare missense variants (R2034Q, L2118V, 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 @GENE$ 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 @GENE$ 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 UBQLN2 gene.

SPG11;41614

UBQLN2;81830

A579T;tmVar:c|SUB|A|579|T;HGVS:c.579A>T;VariantGroup:8;CorrespondingGene:3798;RS#:760135493

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

0no label

Five anencephaly cases carried rare or novel CELSR1 missense variants, three of whom carried additional rare potentially damaging PCP variants: 01F377 (@GENE$ c.6362G>A and PRICKLE4 c.730C>G), 2F07 (CELSR1 @VARIANT$ and DVL3 c.1622C>T), 618F05 (CELSR1 c.8282C>T and SCRIB c.3979G>A). One patient (f93-80) had a novel PTK7 missense variant (c.655A>G) with a rare CELSR2 missense variant (c.1892C>T). Three patients carried missense variants both in FZD and other PCP-associated genes: 01F552 (FZD6 @VARIANT$ and CELSR2 c.3800A>G), 335F07 (@GENE$ 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).

CELSR1;7665

FZD6;2617

c.8807C>T;tmVar:c|SUB|C|8807|T;HGVS:c.8807C>T;VariantGroup:24;CorrespondingGene:9620;RS#:201509338;CA#:10292625

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

0no label

Three rare missense variants (@VARIANT$, L2118V, and E2003D) of the SPG11 gene were found. The high detection rate of missense variants of this gene is probably due to the large size of the coding region; therefore, we suggest that these SPG11 variants are unlikely to be deleterious. Variants in the @GENE$ 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$,) and a novel variant (Q84H) were found in the @GENE$ gene.

SPG11;41614

UBQLN2;81830

R2034Q;tmVar:p|SUB|R|2034|Q;HGVS:p.R2034Q;VariantGroup:26;CorrespondingGene:80208;RS#:750101301;CA#:7534261

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

0no label

The p.Arg153Cys (c.457C>T) mutation was found in exon 3 of EDA, it results in the substitution of @VARIANT$. Moreover, a heterozygous @VARIANT$ (c.637G>A) mutation was detected in exon 3 of @GENE$, this leads to the substitution of Gly at residue 213 to Ser. Sequence analyses revealed that both mutant alleles were from his mother (Fig. 2D), who had a very mild phenotype of isolated tooth agenesis. His father did not have mutations in either of these genes. "S3" is a 14-year-old girl who had the typical clinical characteristics of HED: sparse hair, 26 missing permanent teeth, hypohidrosis, dry skin, and eczema on her body, but no plantar hyperkeratosis or nail abnormalities (Table 1). The heterozygous p.Arg156Cys (c.466C>T) mutation was found in exon 3 of @GENE$, it results in the substitution of Arg at residue 156 to Cys.

WNT10A;22525

EDA;1896

Arg at residue 153 to Cys;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

DISCUSSION We present a Chinese family with PFBC in which the previously reported heterozygous mutation c.1787A>G (@VARIANT$) in @GENE$ and the SNP (rs544478083) c.317G>C (p.Arg106Pro) in @GENE$ 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 PDGFRB @VARIANT$ (p.Arg106Pro) variant showed very slight calcification and was clinically asymptomatic.

SLC20A2;68531

PDGFRB;1960

p.His596Arg;tmVar:p|SUB|H|596|R;HGVS:p.H596R;VariantGroup:2;CorrespondingGene:6575

c.317G>C;tmVar:c|SUB|G|317|C;HGVS:c.317G>C;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

0no label

However, none of these signs were evident from metabolic work of the patient with PHKA1 @VARIANT$, thus ruling out pathogenic significance of this variant. Pathogenic effects of GBE1 D413N and NDUFS8 @VARIANT$ variants remain unknown. It is important to note that these variants changed amino acids that are highly conserved in species from human down to bacteria (data not shown). Because dominant mutations in @GENE$ and @GENE$ are associated with MHS, we evaluated MH diagnostic test results from clinical history of these two subjects.

RYR1;68069

CACNA1S;37257

L718F;tmVar:p|SUB|L|718|F;HGVS:p.L718F;VariantGroup:7;CorrespondingGene:5256;RS#:931442658;CA#:327030635

I126V;tmVar:p|SUB|I|126|V;HGVS:p.I126V;VariantGroup:0;CorrespondingGene:4728;RS#:1267270290

0no label

(A) In the @GENE$ exon 4 and exon 9, the arrows indicate the nucleotide substitution, c.475A > G and c.1051A > G, consisting, respectively, in the amino acid substitutions, @VARIANT$ (A/G heterozygous patient and mother, A/A wild type father) and R351G; (B) in the NOD2 exon 9 sequence, the c.2857 A > G substitution consisted in an amino acid substitution, @VARIANT$ (A/G heterozygous patient and mother, A/A wild-type father). Bioinformatics analysis results. (A) Multiple alignment of the amino acid sequence of @GENE$ protein in seven species showed that this is a conserved region; (B) PolyPhen2 (Polymorphism Phenotyping v.2) analysis predicting the probably damaging impact of the K953E substitution with a score of 0.999.

IL10RA;1196

NOD2;11156

S159G;tmVar:p|SUB|S|159|G;HGVS:p.S159G;VariantGroup:0;CorrespondingGene:3587;RS#:8178561

K953E;tmVar:p|SUB|K|953|E;HGVS:p.K953E;VariantGroup:0;CorrespondingGene:64127;RS#:8178561

0no label

To investigate the role of @GENE$ variations along with GJB2 mutations for a possible combinatory allelic disease inheritance, we have screened patients with heterozygous @GENE$ 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 (@VARIANT$/N166S, 235delC/A194T and 299delAT/@VARIANT$).

GJB3;7338

GJB2;2975

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

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

0no label

The SLC20A2 c.1787A>G (@VARIANT$) variant detected in our study has been reported to cause brain calcification without clinical manifestations due to @GENE$ dysfunction, which probably results in the accumulation of Pi in affected brain regions (Guo et al., 2019). In addition, the @GENE$ @VARIANT$ (p.Arg106Pro) variant, which may destroy the integrity of the BBB, leading to the transfer of Pi from blood vessels into the brain and further promote the accumulation of Pi in affected brain regions.

PiT2;68531

PDGFRB;1960

p.His596Arg;tmVar:p|SUB|H|596|R;HGVS:p.H596R;VariantGroup:2;CorrespondingGene:6575

c.317G>C;tmVar:c|SUB|G|317|C;HGVS:c.317G>C;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

0no label

In this study, we sequenced complete exome in two affected individuals and identified candidate variants in @GENE$ (@VARIANT$), SNAI2 (@VARIANT$) and C2orf74 (c.101T>G) genes. Variant in @GENE$ is not segregating with the disease phenotype therefore it was excluded as an underlying cause of WS2 in the family.

MITF;4892

SNAI2;31127

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

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

0no label

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 @VARIANT$ of the S100A3 protein (c.229C>T; p.R77C) and d) the c.238-241delATTG (@VARIANT$) in S100A13. Mutation name is based on the full-length @GENE$ (NM_002960) and @GENE$ (NM_001024210) transcripts.

S100A3;2223

S100A13;7523

arginine residue to cysteine at position 77;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

Patient P0432 has a @VARIANT$ (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. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (c.46C>G; @VARIANT$) and @GENE$ (c.9921T>G).

CDH23;11142

USH2A;66151

c.4030_4037delATGGCTGG;tmVar:c|DEL|4030_4037|ATGGCTGG;HGVS:c.4030_4037delATGGCTGG;VariantGroup:216;CorrespondingGene:7399

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

0no label

The nucleotide sequence showed a T deletion at nucleotide 252 (c.252DelT) of the coding sequence in exon 1 of @GENE$; this leads to a frame shift from residue 84 and a premature @VARIANT$. 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 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 p.Gly213Ser (@VARIANT$) mutation was detected in exon 3 of WNT10A, 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.637G>A;tmVar:c|SUB|G|637|A;HGVS:c.637G>A;VariantGroup:4;CorrespondingGene:80326;RS#:147680216;CA#:211313

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 (@VARIANT$; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; p.Arg203Cys) and @GENE$ (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients. Variant in @GENE$ (c.607C>T; @VARIANT$) gene is rare in population and is probably damaging and deleterious as predicted by PolyPhen2 and SIFT, respectively.

TYRO3;4585

SNAI3;8500

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

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

0no label

Four cases were identified to carry SQSTM1 variants: the P392L in two cases and the @VARIANT$ and R393Q in single patients. All three alterations are located within the C-terminal ubiquitin-associated (UBA) end of the sequestome 1 protein. Variants of the SQSTM1 gene were originally reported in Paget's disease of bone. However, recent publications suggest a link between SQSTM1 variants and ALS/FTD. The P392L and R393Q variants are known variants reported by other study groups. Interestingly, the patient (#73u) carrying the novel E389Q variant was also diagnosed with Paget's disease of bone. In addition, this patient also carried a variant of unknown significance (@VARIANT$) in the @GENE$ gene in heterozygous form. This case exemplifies the relevant observation of phenotypic pleiotropy and highlights the complexity of the phenotype-genotype correlation. Variants in the @GENE$ gene has been previously linked to autosomal dominant hereditary spastic paraparesis (SPG10) and to Charcot-Marie-Tooth disease type 2 (CMT2).

SIGMAR1;39965

KIF5A;55861

E389Q;tmVar:p|SUB|E|389|Q;HGVS:p.E389Q;VariantGroup:24;CorrespondingGene:8878;RS#:1391182750

I42R;tmVar:p|SUB|I|42|R;HGVS:p.I42R;VariantGroup:1;CorrespondingGene:10280;RS#:1206984068

0no label

Analysis of the entire coding region of the @GENE$ gene revealed the presence of two different missense mutations (@VARIANT$ and A194T) occurring in compound heterozygosity along with the 235delC and 299delAT of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and 299delAT/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 asparagine into serine substitution in codon 166 (N166S) and for the @VARIANT$ of @GENE$ (Fig. 1b, d).

Cx31;7338

GJB2;2975

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 investigate the role of GJB3 variations along with @GENE$ 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 299delAT/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 @GENE$, resulting in an @VARIANT$ (N166S) and for the @VARIANT$ of GJB2 (Fig. 1b, d).

GJB2;2975

GJB3;7338

asparagine into serine substitution in codon 166;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

In the present study, we found two variants: the @VARIANT$ variant in two patients and the A579T variant in one case, with both variants located within the coiled-coil domain (amino acid positions 331-906) of the protein, which is not in line with previous findings. Without additional functional evidence, the pathogenicity of these variants is uncertain. Three rare missense variants (R2034Q, L2118V, 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 @GENE$ 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 @GENE$ 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.

SPG11;41614

UBQLN2;81830

E758K;tmVar:p|SUB|E|758|K;HGVS:p.E758K;VariantGroup:23;CorrespondingGene:3798;RS#:140281678;CA#:6653063

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

0no label

The @VARIANT$ (c.1045G>A) mutation in exon 9 of @GENE$ and heterozygous p.Arg171Cys (c.511C>T) mutation in exon 3 of @GENE$ were detected. These mutations were not found in his father's genome, but because his mother's DNA sample was unavailable, the origin of the mutant alleles was not clear (Fig. 2F). All novel mutations that were identified in this study were not found in the normal controls. Protein structure analysis The results of protein structure analyses of WNT10A are shown in Figure 3. R171 and @VARIANT$ are conserved residues through these organisms and located on conserved 2D fragments.

EDA;1896

WNT10A;22525

p.Ala349Thr;tmVar:p|SUB|A|349|T;HGVS:p.A349T;VariantGroup:2;CorrespondingGene:1896;RS#:132630317;CA#:255657

G213;tmVar:c|Allele|G|213;VariantGroup:4;CorrespondingGene:80326;RS#:147680216

0no label

We report digenic variants in SCRIB and @GENE$ associated with NTDs in addition to @GENE$ and CELSR1 heterozygous variants in additional NTD cases. The combinatorial variation of PTK7 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.

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

Notably, the patients carrying the p.T688A and p.I400V mutations, and three patients carrying the p.V435I mutation also carry, in heterozygous state, p.Y217D, @VARIANT$ (two patients), @VARIANT$, and p.G687N pathogenic mutations in @GENE$, @GENE$, PROK2, and FGFR1, respectively (Table 1), which further substantiates the digenic/oligogenic mode of inheritance of KS.

KAL1;55445

PROKR2;16368

p.R268C;tmVar:p|SUB|R|268|C;HGVS:p.R268C;VariantGroup:8;CorrespondingGene:128674;RS#:78861628;CA#:9754278

p.H70fsX5;tmVar:p|FS|H|70||5;HGVS:p.H70fsX5;VariantGroup:9;CorrespondingGene:60675

0no label

Here we present a patient with severe, progressive neonatal HCM, elevated urinary catecholamine metabolites, and dysmorphic features in whom we identified a known LEOPARD syndrome-associated @GENE$ mutation (c.1403 C > T; @VARIANT$) and a novel, potentially pathogenic missense @GENE$ variant (c.1018 C > T; @VARIANT$) replacing a rigid nonpolar imino acid with a polar amino acid at a highly conserved position.

PTPN11;2122

SOS1;4117

p.T468M;tmVar:p|SUB|T|468|M;HGVS:p.T468M;VariantGroup:6;CorrespondingGene:5781;RS#:121918457;CA#:220134

p.P340S;tmVar:p|SUB|P|340|S;HGVS:p.P340S;VariantGroup:2;CorrespondingGene:6654;RS#:190222208;CA#:1624660

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On the other hand, two missense mutations of the EPHA2 gene were identified in two families, SLC26A4: c.1300G>A (p.434A>T), EPHA2: c.1063G>A (p.G355R) and @GENE$: c.1229C>A (@VARIANT$), @GENE$: @VARIANT$ (p.T511M) (Fig. 6a, b).

SLC26A4;20132

EPHA2;20929

p.410T>M;tmVar:p|SUB|T|410|M;HGVS:p.T410M;VariantGroup:9;CorrespondingGene:5172;RS#:111033220;CA#:261403

c.1532C>T;tmVar:c|SUB|C|1532|T;HGVS:c.1532C>T;VariantGroup:5;CorrespondingGene:1969;RS#:55747232;CA#:625151

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(D, E) A total of 293 T-cells were transfected with Flag-tagged WT or mutant FLNB (p.@VARIANT$, p.A2282T) vector plasmids and myc-tagged WT or mutant TTC26 (p.R297C, @VARIANT$). Then, communoprecipitation assays were conducted. Western blot images are representative of n=3 experiments. AIS, adolescent idiopathic scoliosis; WT, wild type. To investigate the protein-protein interactions, we focused on AIS trios with multiple variants. We found that patients in two AIS trios (trios 22 and 27) carried variants in both the @GENE$ and @GENE$ genes (figure 1).

FLNB;37480

TTC26;11786

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

p.R50C;tmVar:p|SUB|R|50|C;HGVS:p.R50C;VariantGroup:21;CorrespondingGene:79989;RS#:143880653;CA#:4508058

0no label

In patient AVM028, one novel heterozygous VUS (@VARIANT$ [p.His736Arg]) in RASA1 inherited from the father and one likely pathogenic de novo novel heterozygous variant (@VARIANT$ [p.Leu104Pro]) in TIMP3 were identified (online supplementary table S2). While TIMP3 blocks VEGF/@GENE$ signalling, @GENE$ modulates differentiation and proliferation of blood vessel endothelial cells downstream of VEGF (figure 3).

VEGFR2;55639

RASA1;2168

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

c.311T>C;tmVar:c|SUB|T|311|C;HGVS:c.311T>C;VariantGroup:7;CorrespondingGene:5783;RS#:1290872293

0no label

@GENE$ functions as a coreceptor that enhances VEGF/@GENE$ binding to stimulate VEGF signalling. In this case, both the TGF-beta and VEGF signalling pathways could be affected, potentially causing a more severe downstream effect than would occur with variants in only one of the pathways, with the mutations synergising to lead to BAVM. In patient AVM028, one novel heterozygous VUS (c.2207A>G [@VARIANT$]) in RASA1 inherited from the father and one likely pathogenic de novo novel heterozygous variant (@VARIANT$ [p.Leu104Pro]) in TIMP3 were identified (online supplementary table S2).

SCUBE2;36383

VEGFR2;55639

p.His736Arg;tmVar:p|SUB|H|736|R;HGVS:p.H736R;VariantGroup:6;CorrespondingGene:5921;RS#:1403332745

c.311T>C;tmVar:c|SUB|T|311|C;HGVS:c.311T>C;VariantGroup:7;CorrespondingGene:5783;RS#:1290872293

0no label

Both homozygous and compound heterozygous variants in the @GENE$ gene have been described as causative for juvenile ALS. The G1177X 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. @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, @VARIANT$) were detected. The P11S variant affects the b isoform of the MATR3 protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms. Further evidence is required to elucidate the mechanism of pathogenicity of these alterations. We discovered several variants in ALS candidate and risk genes. In a patient with LMN-dominant ALS with slow progression, we found two novel variants (T2583I and @VARIANT$) in the DYNC1H1 gene.

ALS2;23264

MATR3;7830

S275N;tmVar:p|SUB|S|275|N;HGVS:p.S275N;VariantGroup:9;CorrespondingGene:80208;RS#:995711809

G4290R;tmVar:p|SUB|G|4290|R;HGVS:p.G4290R;VariantGroup:27;CorrespondingGene:1778;RS#:748643448;CA#:7354051

0no label

Moreover, mutations in residues close to @VARIANT$ and A194 identified in the families reported here, namely, M163L, R165W, F191L, and A197S in Cx26 as well as @VARIANT$, S198F and G199R in Cx32, have been reported previously in patients with hearing impairment. Interestingly, mutations identified in patients with the skin disease erythrokeratoderma variabilis (EKV) were located within all the protein domains of the Cx31 gene except for the EC2 and TM4 domains, which are main domains for deafness mutations. This correlation between location of mutations and phenotypes, together with the identification of pathological mutations associated with hearing loss in the same region of the EC2 and TM4 domains in these three connexin genes (Cx26, Cx31, and @GENE$) suggested that the EC2 and TM4 domains are important to the function of the Cx31 protein in the inner ear and plays a vital role in forming connexons in the cells of the inner ear. In the present study, we have shown that the missense N166S and A194T mutations in @GENE$ acts in a recessive manner in three unrelated Chinese patients.

Cx32;137

GJB3;7338

N166;tmVar:p|Allele|N|166;VariantGroup:0;CorrespondingGene:2707;RS#:121908851

F193C;tmVar:p|SUB|F|193|C;HGVS:p.F193C;VariantGroup:15;CorrespondingGene:2706

0no label

KCNH2-@VARIANT$ affects also the synchronization between depolarization and repolarization and so increases the risk of cardiac mortality. Therefore, it is a genetic modifier candidate. Finally, as reported in population studies, @GENE$-p.G38S is associated with heart failure, atrial fibrillation, abnormal cardiac repolarization, and an increased risk of ventricular arrhythmia. Nevertheless, in vitro studies demonstrated that the KCNE1-@VARIANT$ variant causes only a mild reduction of the delayed rectifier K+ currents. Therefore, G38S could be a genetic modifier, but the evidence available does not suggest it has an overt effect on the function of the KCNQ1 and @GENE$ channels.

KCNE1;3753

KCNH2;201

p.K897T;tmVar:p|SUB|K|897|T;HGVS:p.K897T;VariantGroup:0;CorrespondingGene:3757;RS#:1805123;CA#:7162

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

0no label

A PCR amplicon containing @GENE$ exons 2 and 3 was partially sequenced and revealed heterozygosity for an intron 2 polymorphism (@VARIANT$), thereby indicating the presence of two copies of each exon and excluding the possibility of exon deletion as the second mutation in this patient. The screening of other genes related to the hypothalamic-pituitary-gonadal axis, in this patient, revealed an additional heterozygous missense mutation (c.[238C > T];[=]) (@VARIANT$) in the @GENE$ gene.

GNRHR;350

PROKR2;16368

rs373270328;tmVar:rs373270328;VariantGroup:0;CorrespondingGene:2798;RS#:373270328

p.Arg80Cys;tmVar:p|SUB|R|80|C;HGVS:p.R80C;VariantGroup:4;CorrespondingGene:128674;RS#:774093318;CA#:9754400

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Notably, the patients carrying the @VARIANT$ and p.I400V mutations, and three patients carrying the p.V435I mutation also carry, in heterozygous state, p.Y217D, p.R268C (two patients), @VARIANT$, and p.G687N pathogenic mutations in KAL1, @GENE$, @GENE$, and FGFR1, respectively (Table 1), which further substantiates the digenic/oligogenic mode of inheritance of KS.

PROKR2;16368

PROK2;9268

p.T688A;tmVar:p|SUB|T|688|A;HGVS:p.T688A;VariantGroup:0;CorrespondingGene:2260;RS#:876661335

p.H70fsX5;tmVar:p|FS|H|70||5;HGVS:p.H70fsX5;VariantGroup:9;CorrespondingGene:60675

0no label

Pedigree and segregation of the mutations in @GENE$ and GJB3 The deaf proband is indicated by an arrow. GJB2/GJB3 genotypes are given below the respective pedigrees symbol (a, f and k). Direct sequence analysis showing the 235delC mutation (b and g) and wild type (WT) allele (c and h) of GJB2. Direct sequence analysis showing the @VARIANT$ mutation (l) and wild type (WT) allele (m) of GJB2. Direct sequence analysis showing the @VARIANT$ (N166S) mutation (d) and WT allele (e) of @GENE$. Direct sequence analysis showing the 580G>A (A194T) mutation (i and n) and WT allele (j and o) of GJB3.

GJB2;2975

GJB3;7338

299-300delAT;tmVar:c|DEL|299_300|AT;HGVS:c.299_300delAT;VariantGroup:2;CorrespondingGene:2706;RS#:111033204

497A>G;tmVar:c|SUB|A|497|G;HGVS:c.497A>G;VariantGroup:0;CorrespondingGene:2707;RS#:121908851;CA#:118311

0no label

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 @GENE$ 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 @GENE$ 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, @VARIANT$/@VARIANT$ and 299delAT/A194T).

Cx26;2975

Cx31;7338

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

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

0no label

The @VARIANT$ and @VARIANT$ variants affect the conserved central coiled-coil rod domain of the protein mediating dimerization; therefore, we suggest their potential deleterious effect on the protein. In the individual carrying the P505L NEFH variant, an additional novel alteration (C335R) was detected in the GRN gene. Loss-of-function @GENE$ variants are primarily considered to cause frontotemporal lobar degeneration, but there is evidence that missense GRN variants are also linked to the pathogenesis of ALS. The novel GRN variant reported in this study results in a cysteine-to-arginine change in the cysteine-rich granulin A domain. Four cases were identified to carry SQSTM1 variants: the P392L in two cases and the E389Q and R393Q in single patients. All three alterations are located within the C-terminal ubiquitin-associated (UBA) end of the sequestome 1 protein. Variants of the @GENE$ gene were originally reported in Paget's disease of bone.

GRN;1577

SQSTM1;31202

T338I;tmVar:p|SUB|T|338|I;HGVS:p.T338I;VariantGroup:5;CorrespondingGene:4744;RS#:774252076;CA#:10174087

R148P;tmVar:p|SUB|R|148|P;HGVS:p.R148P;VariantGroup:14;CorrespondingGene:2521;RS#:773655049

0no label

Twenty-two rare variants were shared by the three patients (Tables 1 and S1), including variants in the @GENE$ (NM_000179.2: c.3299C > T, @VARIANT$) and @GENE$ (NM_001128425.1: c.536A > G, @VARIANT$) genes, while the other 20 genes could not be clearly linked to cancer predisposition.

MSH6;149

MUTYH;8156

p.Thr1100Met;tmVar:p|SUB|T|1100|M;HGVS:p.T1100M;VariantGroup:4;CorrespondingGene:2956;RS#:63750442;CA#:12473

p.Tyr179Cys;tmVar:p|SUB|Y|179|C;HGVS:p.Y179C;VariantGroup:15;CorrespondingGene:4595;RS#:145090475

11

Variants in all known WS candidate genes (EDN3, EDNRB, @GENE$, PAX3, SOX10, SNAI2, and @GENE$) were searched and a novel rare heterozygous deletion mutation (@VARIANT$; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; p.Arg203Cys) and TYRO3 (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

MITF;4892

TYRO3;4585

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

2.3. Functional Consequences of the @GENE$-p.R583H and @GENE$-p.C108Y Variants To investigate the functional consequences of KCNQ1-@VARIANT$ and KCNH2-@VARIANT$, we performed whole cell patch clamp experiments in transiently transfected CHO-K1 cells.

KCNQ1;85014

KCNH2;201

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

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

0no label

Its numerous domains allow @GENE$ to serve as a frame for multiprotein complexes and regulator of ubiquitinated protein turnover. SQSTM1 mutations have been linked with a spectrum of phenotypes, including Paget disease of bone (PDB), ALS, FTD, and MRV. Hence, SQSTM1 mutations can lead to a multisystem proteinopathy although with incomplete penetrance. 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 (@VARIANT$, p.Asn357Ser) by Evila et al.. Evila et al.'s study reported also an additional sporadic MRV case carrying the same TIA1 variant but a different SQSTM1 mutation (p.Pro392Leu), which is known to cause PDB, ALS, and FTD, but the patient's phenotype was not illustrated.

SQSTM1;31202

TIA1;20692

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

c.1070A>G;tmVar:c|SUB|A|1070|G;HGVS:c.1070A>G;VariantGroup:5;CorrespondingGene:7072;RS#:116621885;CA#:1697407

0no label

The @VARIANT$ variant affects the b isoform of the @GENE$ protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms. Further evidence is required to elucidate the mechanism of pathogenicity of these alterations. We discovered several variants in ALS candidate and risk genes. In a patient with LMN-dominant ALS with slow progression, we found two novel variants (@VARIANT$ and G4290R) in the @GENE$ gene.

MATR3;7830

DYNC1H1;1053

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

T2583I;tmVar:p|SUB|T|2583|I;HGVS:p.T2583I;VariantGroup:31;CorrespondingGene:1778

0no label

To investigate the role of @GENE$ variations along with GJB2 mutations for a possible combinatory allelic disease inheritance, we have screened patients with heterozygous @GENE$ 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 (@VARIANT$/@VARIANT$, 235delC/A194T and 299delAT/A194T).

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

11

This analysis indicated that the @GENE$ variant @VARIANT$ (rs138172448), which results in a p.Val555Ile change, and the @GENE$ gene variant @VARIANT$ (rs144901249), which results in a p.Thr219Ile change, are both predicted to be damaging.

CAPN3;52

DES;56469

c.1663G>A;tmVar:c|SUB|G|1663|A;HGVS:c.1663G>A;VariantGroup:2;CorrespondingGene:825;RS#:138172448;CA#:7511461

c.656C>T;tmVar:c|SUB|C|656|T;HGVS:c.656C>T;VariantGroup:3;CorrespondingGene:1674;RS#:144901249;CA#:2125118

0no label

Except for the @GENE$ gene variant [p.(Glu436Lys)], mutations identified in DUSP6, ANOS1, DCC, PLXNA1, and PROP1 genes were carried by HH1 family cases (HH1, HH1F, and HH1P) and involved in pathogenic digenic combinations with the @GENE$ gene variant [p.(@VARIANT$)]. Such findings bring into question their involvement in disease expression in HH12. The SEMA7A variant [p.(@VARIANT$)] was predicted as VUS by Varsome.

SEMA7A;2678

DUSP6;55621

Val114Leu;tmVar:p|SUB|V|114|L;HGVS:p.V114L;VariantGroup:5;CorrespondingGene:1848;RS#:2279574;CA#:6714072

Glu436Lys;tmVar:p|SUB|E|436|K;HGVS:p.E436K;VariantGroup:8;CorrespondingGene:54756;RS#:1411341050

0no label

We have excluded the possibility that mutations in exon 1 of @GENE$ and the deletion of @GENE$ are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S 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/@VARIANT$).

GJB2;2975

GJB6;4936

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

0no label

Results Cosegregating deleterious variants (GRCH37/hg19) in @GENE$ (NM_001127222.1: @VARIANT$, p.Pro2421Val), REEP4 (NM_025232.3: c.109C>T, p.Arg37Trp), TOR2A (NM_130459.3: @VARIANT$, p.Arg190Cys), and @GENE$ (NM_005173.3: c.1966C>T, p.Arg656Cys) were identified in four independent multigenerational pedigrees.

CACNA1A;56383

ATP2A3;69131

c.7261_7262delinsGT;tmVar:c|INDEL|7261_7262|GT;HGVS:c.7261_7262delinsGT;VariantGroup:32;CorrespondingGene:773

c.568C>T;tmVar:c|SUB|C|568|T;HGVS:c.568C>T;VariantGroup:12;CorrespondingGene:27433;RS#:376074923;CA#:5250615

0no label

The proband's father with the @GENE$ c.1787A>G (@VARIANT$) mutation showed obvious brain calcification but was clinically asymptomatic. The proband's mother with the @GENE$ @VARIANT$ (p.Arg106Pro) variant showed very slight calcification and was clinically asymptomatic.

SLC20A2;68531

PDGFRB;1960

p.His596Arg;tmVar:p|SUB|H|596|R;HGVS:p.H596R;VariantGroup:2;CorrespondingGene:6575

c.317G>C;tmVar:c|SUB|G|317|C;HGVS:c.317G>C;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

11

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 @VARIANT$ of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and 299delAT/@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

0no label

We found that @GENE$-@VARIANT$ was not associated with a severe functional impairment, whereas KCNH2-p.C108Y, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type. Notably, the common variants KCNH2-p.K897T and KCNE1-p.G38S were previously reported to produce more severe phenotypes when combined with disease-causing alleles. Our results indicate that the novel KCNH2-C108Y variant can be a pathogenic LQTS mutation, whereas KCNQ1-p.R583H, KCNH2-p.K897T, and @GENE$-@VARIANT$ could be LQTS modifiers.

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

0no label

(C) The sequence of the @VARIANT$ variant is well-conserved from humans to tunicates. (D) SH175-389 harbored a monoallelic p.V193E variant of GJB2 and a monoallelic @VARIANT$ variant of GJB3. DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = @GENE$, GJB3 = gap junction protein beta 3, @GENE$ = gap junction protein beta 6, MITF = microphthalmia-associated transcription factor.

gap junction protein beta 2;2975

GJB6;4936

p.R341C;tmVar:p|SUB|R|341|C;HGVS:p.R341C;VariantGroup:7;CorrespondingGene:161497;RS#:1359505251

p.A194T;tmVar:p|SUB|A|194|T;HGVS:p.A194T;VariantGroup:18;CorrespondingGene:2707;RS#:117385606;CA#:118313

0no label

In addition, 2 genes presented variants in 3 patients: @GENE$ (patients 6, 7 and 8) and @GENE$ (patients 1, 4 and 8). Furthermore, RIPK4 presented 2 variants in patient 1. Finally, BNC2 variant c.1868C>A:@VARIANT$ (MAF = 0.002) was detected in 2 patients (patient 1 and 7) and MAML3 variant c.881A>G:@VARIANT$ (MAF = 0.0028) in patients 7 and 8 ( Table 2 ).

MAML3;41284

NOTCH1;32049

p.(Pro623His);tmVar:p|SUB|P|623|H;HGVS:p.P623H;VariantGroup:11;CorrespondingGene:54796;RS#:114596065;CA#:204322

p.(Asn294Ser);tmVar:p|SUB|N|294|S;HGVS:p.N294S;VariantGroup:16;CorrespondingGene:55534;RS#:115966590;CA#:3085269

0no label

However, none of these signs were evident from metabolic work of the patient with PHKA1 @VARIANT$, thus ruling out pathogenic significance of this variant. Pathogenic effects of @GENE$ @VARIANT$ and @GENE$ I126V variants remain unknown.

GBE1;129

NDUFS8;1867

L718F;tmVar:p|SUB|L|718|F;HGVS:p.L718F;VariantGroup:7;CorrespondingGene:5256;RS#:931442658;CA#:327030635

D413N;tmVar:p|SUB|D|413|N;HGVS:p.D413N;VariantGroup:8;CorrespondingGene:2632;RS#:752711257

0no label

18 , 19 This gene codes for several isoforms, including the ubiquitously expressed p200 CUX1, which, among other functions, has been shown to stimulate the repair of oxidized DNA bases by @GENE$. 20 The identified @GENE$ (NM_001202543: @VARIANT$, p.Ser480Gly) variant, however, was classified as likely benign by the Franklin variant classification tool. 21 Additional gene reportedly linked to tumorigenesis include RYR3, 22 EBNA1BP2, 23 TRIP6, 24 and CAPN9. 25 The RYR3 (NM_001036: c.7812C > G, p.Asn2604Lys) and EBNA1BP2 (NM_001159936: c.1034A > T, p.Asn345Ile) variants were classified as likely benign and benign, respectively, while the TRIP6 (NM_003302: c.822G > C, p.Glu274Asp) and the CAPN9 (NM_006615: @VARIANT$, p.Ala19Ser) variants were classified as VUS.

OGG1;1909

CUX1;22551

c.1438A > G;tmVar:c|SUB|A|1438|G;HGVS:c.1438A>G;VariantGroup:2;CorrespondingGene:1523;RS#:147066011;CA#:4410849

c.55G > T;tmVar:c|SUB|G|55|T;HGVS:c.55G>T;VariantGroup:17;CorrespondingGene:10753;RS#:147360179;CA#:1448452

0no label

DISCUSSION We present a Chinese family with PFBC in which the previously reported heterozygous mutation c.1787A>G (p.His596Arg) in @GENE$ and the SNP (rs544478083) @VARIANT$ (p.Arg106Pro) in @GENE$ were identified. The proband's father with the SLC20A2 c.1787A>G (@VARIANT$) mutation showed obvious brain calcification but was clinically asymptomatic.

SLC20A2;68531

PDGFRB;1960

c.317G>C;tmVar:c|SUB|G|317|C;HGVS:c.317G>C;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

p.His596Arg;tmVar:p|SUB|H|596|R;HGVS:p.H596R;VariantGroup:2;CorrespondingGene:6575

0no label

(A) The EDA mutation @VARIANT$ and WNT10A mutation c.511C>T were found in patient N1, who inherited the mutant allele from his mother. (B) The @GENE$ mutation c.936C>G and @GENE$ mutation @VARIANT$ were found in patient N2, who also inherited the mutant allele from his mother.

EDA;1896

WNT10A;22525

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

(b) A sequence chromatogram showing the @GENE$ (c.253C>T;@VARIANT$) mutation. (c) A sequence chromatogram showing the @GENE$ (c.1306A>G;@VARIANT$) mutation.

PROKR2;16368

WDR11;41229

p.R85C;tmVar:p|SUB|R|85|C;HGVS:p.R85C;VariantGroup:1;CorrespondingGene:128674;RS#:74315418

p.I436V;tmVar:p|SUB|I|436|V;HGVS:p.I436V;VariantGroup:3;CorrespondingGene:55717;RS#:34602786;CA#:5719694

0no label

Merged images showing pro-COL1A1 colocalization with @GENE$ in wild-type (C); SEC23AM400I/+ heterozygous (F); SEC23AM400I/+ @GENE$R334C/+ double heterozygous (I); and SEC23AM400I/@VARIANT$ MAN1B1R334C/@VARIANT$ double-homozygous (L) fibroblasts.

TGN38;136490

MAN1B1;5230

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

The @VARIANT$ (p.R77C) variant in S100A3 and c.238-241delATTG (@VARIANT$) mutation in S100A13 also segregated fully with ILD in Families 1B and 2. Haplotype analysis Haplotype analysis carried out using eight markers (four microsatellite markers flanking @GENE$, @GENE$ and three further intragenic markers) (supplementary figure S1a) confirmed that all affected individuals from both families shared a specific disease haplotype on both chromosomes that was not present in the unaffected individuals, suggesting a shared extended haplotype from a common founder.

S100A3;2223

S100A13;7523

c.229C>T;tmVar:c|SUB|C|229|T;HGVS:c.229C>T;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

Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: @VARIANT$ and @VARIANT$; @GENE$: p.(P179T)). In summary, the increased frequency in PCG patients of rare @GENE$ and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.

PITX2;55454

FOXC2;21091

p.(C498R);tmVar:p|SUB|C|498|R;HGVS:p.C498R;VariantGroup:1;CorrespondingGene:103752587;RS#:61753346;CA#:8218498

p.(H395N);tmVar:p|SUB|H|395|N;HGVS:p.H395N;VariantGroup:8;CorrespondingGene:2303

0no label

Loss-of-function @GENE$ variants are primarily considered to cause frontotemporal lobar degeneration, but there is evidence that missense GRN variants are also linked to the pathogenesis of ALS. The novel GRN variant reported in this study results in a cysteine-to-arginine change in the cysteine-rich granulin A domain. Four cases were identified to carry SQSTM1 variants: the @VARIANT$ in two cases and the E389Q and R393Q in single patients. All three alterations are located within the C-terminal ubiquitin-associated (UBA) end of the sequestome 1 protein. Variants of the SQSTM1 gene were originally reported in Paget's disease of bone. However, recent publications suggest a link between @GENE$ variants and ALS/FTD. The P392L and R393Q variants are known variants reported by other study groups. Interestingly, the patient (#73u) carrying the novel E389Q variant was also diagnosed with Paget's disease of bone. In addition, this patient also carried a variant of unknown significance (@VARIANT$) in the SIGMAR1 gene in heterozygous form.

GRN;1577

SQSTM1;31202

P392L;tmVar:p|SUB|P|392|L;HGVS:p.P392L;VariantGroup:17;CorrespondingGene:8878;RS#:104893941;CA#:203866

I42R;tmVar:p|SUB|I|42|R;HGVS:p.I42R;VariantGroup:1;CorrespondingGene:10280;RS#:1206984068

0no label

Finally, BNC2 variant c.1868C>A:@VARIANT$ (MAF = 0.002) was detected in 2 patients (patient 1 and 7) and MAML3 variant c.881A>G:@VARIANT$ (MAF = 0.0028) in patients 7 and 8 ( Table 2 ). We performed interactome analysis for the identified DSD genes using bioinformatic tools for the analysis of possible gene-protein interactions. The network comprising all genes identified is shown in Figure 1 . Overall, a connection was found for 27 of the 41 genes. MAMLD1 connects directly to MAML1/2/3. Via NOTCH1/2 8 genes are in connection with MAMLD1, namely WNT9A/9B, @GENE$, FGF10, RET, PROP1 and NRP1. Some of these genes are also central nodes for further connections; e.g. GLI3 for EVC, FGF10, GLI2, RIPK4 and EYA1; and RET for PIK3R3 with PTPN11, which also is connected with @GENE$. RIPK4 itself is a central node for ZBTB16, CUL4B, GLI3 and PTPN11.

GLI2/3;2736;2737

RIPK4;10772

p.(Pro623His);tmVar:p|SUB|P|623|H;HGVS:p.P623H;VariantGroup:11;CorrespondingGene:54796;RS#:114596065;CA#:204322

p.(Asn294Ser);tmVar:p|SUB|N|294|S;HGVS:p.N294S;VariantGroup:16;CorrespondingGene:55534;RS#:115966590;CA#:3085269

0no label

To examine whether EphA2 is involved in dysfunction of pendrin caused by these amino acid substitutions, the effect of pendrin L117F, pendrin @VARIANT$, and pendrin @VARIANT$ mutations on EphA2 interaction and internalization was examined. While the amount of co-precipitated pendrin mutants with EphA2 was comparable to that of wild type (wt) pendrin (Fig. 5c, d), the S166N mutant failed to be internalized after ephrin-B2 stimulation (Fig. 5e, f). Taken together, these results further demonstrate that EphA2 could control both pendrin recruitment to the plasma membrane and @GENE$ exclusion from the plasma membrane. @GENE$ mutations in pendred syndrome patients Identification and characterization of EphA2 mutation from hearing loss patients with EVA.

pendrin;20132

EPHA2;20929

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

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

0no label

The T338I and @VARIANT$ variants affect the conserved central coiled-coil rod domain of the protein mediating dimerization; therefore, we suggest their potential deleterious effect on the protein. In the individual carrying the P505L NEFH variant, an additional novel alteration (C335R) was detected in the @GENE$ gene. Loss-of-function GRN variants are primarily considered to cause frontotemporal lobar degeneration, but there is evidence that missense GRN variants are also linked to the pathogenesis of ALS. The novel GRN variant reported in this study results in a cysteine-to-arginine change in the cysteine-rich granulin A domain. Four cases were identified to carry SQSTM1 variants: the P392L in two cases and the E389Q and @VARIANT$ in single patients. All three alterations are located within the C-terminal ubiquitin-associated (UBA) end of the sequestome 1 protein. Variants of the @GENE$ gene were originally reported in Paget's disease of bone.

GRN;1577

SQSTM1;31202

R148P;tmVar:p|SUB|R|148|P;HGVS:p.R148P;VariantGroup:14;CorrespondingGene:2521;RS#:773655049

R393Q;tmVar:p|SUB|R|393|Q;HGVS:p.R393Q;VariantGroup:15;CorrespondingGene:8878;RS#:200551825;CA#:3600852

0no label

Since @VARIANT$ was only partially conserved (Figure S1B) and no in vitro analysis was performed, its functional significance is unknown. No other IHH/KS genes were studied, so digenic disease cannot be excluded. One heterozygous NELF splice mutation (c.1159-14_22del) has been described. However, the only KS individual within the family also had a heterozygous FGFR1 mutation (@VARIANT$), suggesting digenic disease. This NELF deletion was associated with exon 10 skipping, but was not sufficient to cause KS alone. Therefore, no human NELF mutations, supported in vitro, and without mutations in a second gene, have been reported to cause IHH/KS. In the present study, 3/168 (1.8%) of IHH/KS patients had NELF mutations demonstrating impaired function in vitro, which is similar to GNRHR and greater than @GENE$ mutations in nIHH patients. To exclude digenic disease, sequencing of 11 additional genes (CHD7, FGF8, FGFR1, PROK2, PROKR2, @GENE$, TACR3, KAL1, GNRHR, GNRH1, and KISS1R) was performed.

KISS1R;11411

TAC3;7560

Thr478;tmVar:p|Allele|T|478;VariantGroup:0;CorrespondingGene:26012;RS#:121918340

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

0no label

Exome analysis for the proband identified three sequence variants in FTA candidate genes, two in LRP6 (@VARIANT$, c.379T>A, p.Ser127Thr; g.124339A>G, c.3224A>G, p.Asn1075Ser) and one in @GENE$ (g.14574G>C, c.499G>C, @VARIANT$) (Figure 4A). The @GENE$ c.3224A>G mutation is a rare variant with an MAF of 0.0024 in EAS.

WNT10A;22525

LRP6;1747

g.27546T>A;tmVar:g|SUB|T|27546|A;HGVS:g.27546T>A;VariantGroup:1;CorrespondingGene:4040;RS#:17848270;CA#:6455897

p.Glu167Gln;tmVar:p|SUB|E|167|Q;HGVS:p.E167Q;VariantGroup:5;CorrespondingGene:80326;RS#:148714379

0no label

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

TACI;49320

TCF3;2408

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

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$. Additionally, the nucleotide sequence showed a monoallelic C to T transition at nucleotide 511 (@VARIANT$) of the coding sequence in exon 3 of WNT10A, which results in the substitution of Arg at residue 171 to Cys. 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

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

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, p.Met168Arg; g.112084C>G, c.2450C>G, p.Ser817Cys; @VARIANT$, c.4333A>G, p.Met1445Val) and one in @GENE$ (g.14712G>A, @VARIANT$, p.Gly213Ser) (Figure 2A and Figure S2A,B).

LRP6;1747

WNT10A;22525

g.146466A>G;tmVar:g|SUB|A|146466|G;HGVS:g.146466A>G;VariantGroup:6;CorrespondingGene:4040;RS#:761703397

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

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(C) The sequence of the @VARIANT$ variant is well-conserved from humans to tunicates. (D) SH175-389 harbored a monoallelic p.V193E variant of GJB2 and a monoallelic @VARIANT$ variant of GJB3. DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = @GENE$, GJB3 = @GENE$, GJB6 = gap junction protein beta 6, MITF = microphthalmia-associated transcription factor.

gap junction protein beta 2;2975

gap junction protein beta 3;7338

p.R341C;tmVar:p|SUB|R|341|C;HGVS:p.R341C;VariantGroup:7;CorrespondingGene:161497;RS#:1359505251

p.A194T;tmVar:p|SUB|A|194|T;HGVS:p.A194T;VariantGroup:18;CorrespondingGene:2707;RS#:117385606;CA#:118313

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 SNAI3 (c.607C>T; p.Arg203Cys) and @GENE$ (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

MITF;4892

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

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Since @GENE$ is an intraflagellar transport (IFT) protein in cilia, we aimed to identify potential interactions between FLNB and TTC26. Using coimmunoprecipitation assays, we found that the myc-tagged mutant p.R50C and p.R197C TTC26 proteins pulled down the Flag-tagged mutant p.A2282T and p.R566L FLNB proteins, respectively (figure 2D, E). We were also interested in case 98-73, whose twin sister was also diagnosed with AIS (figure 3A). The FLNB missense variant p.R2003H is located in a highly conserved region of the FLNB protein (figure 3B). In silico analyses (figure 3C) indicated that the R2003 residue was solvent accessible and was positioned far from the beta-sheet secondary structure. In the FLNB mutant protein structure, the side-chain of H2003 formed a strong hydrogen bond with E2078. Since OFD1 localises to the base of the cilium, we assumed that FLNB may interact with OFD1. Coimmunoprecipitation analysis indicated an interaction between wild-type @GENE$ and wild-type FLNB, which did not exist between @VARIANT$ FLNB and @VARIANT$ OFD1 (figure 3D).

TTC26;11786

OFD1;2677

p.R2003H;tmVar:p|SUB|R|2003|H;HGVS:p.R2003H;VariantGroup:18;CorrespondingGene:2317;RS#:563096120;CA#:2469226

p.Y437F;tmVar:p|SUB|Y|437|F;HGVS:p.Y437F;VariantGroup:30;CorrespondingGene:8481

0no label

Interestingly, we identified 5 patients (4.8%) with variants in optineurin (OPTN) and TANK-binding kinase 1 (@GENE$) that are predicted to be highly pathogenic, including two double mutants. Case A was a compound heterozygote for mutations in OPTN, carrying the p.Q235* nonsense and @VARIANT$ missense mutation in trans, while case B carried a deletion of @GENE$ exons 13-15 (p.Gly538Glufs*27) and a loss-of-function mutation (@VARIANT$) in TBK1.

TBK1;22742

OPTN;11085

p.A481V;tmVar:p|SUB|A|481|V;HGVS:p.A481V;VariantGroup:1;CorrespondingGene:10133;RS#:377219791;CA#:5410970

p.Arg117*;tmVar:p|SUB|R|117|*;HGVS:p.R117*;VariantGroup:12;CorrespondingGene:5216;RS#:140547520

0no label

Two SALS patients carried multiple ALS-associated variants that are rare in population databases (ANG p.K41I with VAPB @VARIANT$ and @GENE$ p.R408C with SETX @VARIANT$ and @GENE$ p.T14I).

TAF15;131088

SETX;41003

p.M170I;tmVar:p|SUB|M|170|I;HGVS:p.M170I;VariantGroup:45;CorrespondingGene:9217;RS#:143144050;CA#:9924276

p.I2547T;tmVar:p|SUB|I|2547|T;HGVS:p.I2547T;VariantGroup:58;CorrespondingGene:23064;RS#:151117904;CA#:233108

0no label

Deleterious variants in @GENE$ (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 @GENE$,TRPV4,CAPN11,VPS13C,UNC13B,SPTBN4,MYOD1, and MRPL15 were found in two or more independent pedigrees.

HS1BP3;10980

DNAH17;72102

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

Variants in all known WS candidate genes (EDN3, EDNRB, MITF, @GENE$, SOX10, @GENE$, 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 TYRO3 (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients.

PAX3;22494

SNAI2;31127

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

The @GENE$ and @GENE$ variants were excluded based on their frequencies in normal population cohorts. Sanger sequencing of Family 1 showed that both rs138355706 in S100A3 (@VARIANT$, missense causing a p.R77C mutation) and a 4 bp deletion in S100A13 (c.238-241delATTG causing a frameshift @VARIANT$) segregated completely with ILD in Family 1 based upon recessive inheritance (figure 2c and d), were in total linkage disequilibrium, and were present in a cis conformation.

ISG20L2;12814

SETDB1;32157

c.229C>T;tmVar:c|SUB|C|229|T;HGVS:c.229C>T;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

WES demonstrated heterozygous missense mutations in two genes required for pituitary development, a known loss-of-function mutation in PROKR2 (@VARIANT$;p.R85C) inherited from an unaffected mother, and a @GENE$ (@VARIANT$;p.I436V) mutation inherited from an unaffected father. Mutant WDR11 loses its capacity to bind to its functional partner, @GENE$, and to localize to the nucleus.

WDR11;41229

EMX1;55799

c.253C>T;tmVar:c|SUB|C|253|T;HGVS:c.253C>T;VariantGroup:1;CorrespondingGene:128674;RS#:74315418;CA#:259601

c.1306A>G;tmVar:c|SUB|A|1306|G;HGVS:c.1306A>G;VariantGroup:3;CorrespondingGene:55717;RS#:34602786;CA#:5719694

0no label

A male (ID104) was found to have a heterozygous missense variant @VARIANT$ (p.Lys330Met) in @GENE$ and a missense variant @VARIANT$ (p.Leu593Val) in @GENE$. Limited clinical information was available about this male.

EHMT1;11698

SLC9A6;55971

c.989A > T;tmVar:c|SUB|A|989|T;HGVS:c.989A>T;VariantGroup:1;CorrespondingGene:79813;RS#:764291502;CA#:5375151

c.1777C > G;tmVar:c|SUB|C|1777|G;HGVS:c.1777C>G;VariantGroup:7;CorrespondingGene:10479;RS#:149360465

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