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In this study, we sequenced complete exome in two affected individuals and identified candidate variants in @GENE$ (@VARIANT$), @GENE$ (@VARIANT$) and C2orf74 (c.101T>G) genes.

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

11

Twenty-two rare variants were shared by the three patients (Tables 1 and S1), including variants in the MSH6 (NM_000179.2: c.3299C > T, p.Thr1100Met) and @GENE$ (NM_001128425.1: @VARIANT$, p.Tyr179Cys) genes, while the other 20 genes could not be clearly linked to cancer predisposition. The identified @GENE$ variant was classified as a variant of uncertain significance (VUS) in the Leiden Open Variant Database and the InSiGHT DNA Variant Database. 14 , 15 The MUTYH variant is the most common pathogenic variant found in the Netherlands. 2 The digenic inheritance of MSH6 and MUTYH variants. A, The pedigree shows the coinheritance of the monoallelic variants which encode MSH6 @VARIANT$ and MUTYH p.Tyr179Cys in a family affected by colorectal cancer.

MUTYH;8156

MSH6;149

c.536A > G;tmVar:c|SUB|A|536|G;HGVS:c.536A>G;VariantGroup:15;CorrespondingGene:4595;RS#:145090475;CA#:7607273

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

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 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 EDA 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 @GENE$ mutation c.457C>T and @GENE$ 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.

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

0no label

Compared to WT (wild-type) proteins, we found that the ability of GFP-CYP1B1 A115P and GFP-CYP1B1 E229K to immunoprecipitate HA-TEK E103D and HA-@GENE$ @VARIANT$, respectively, was significantly diminished. GFP-@GENE$ @VARIANT$ also exhibited relatively reduced ability to immunoprecipitate HA-TEK I148T (~70%).

TEK;397

CYP1B1;68035

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

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

0no label

After filtering for homozygous nonsynonymous single nucleotide variants (SNVs) within the linkage interval, that were either novel or had either low or unknown minor allele frequency in dbSNP, only three previously described variants, @VARIANT$ in ISG20L2, rs143224912 in @GENE$ and @VARIANT$ in @GENE$, and one novel variant in S100A13, were identified.

SETDB1;32157

S100A3;2223

rs3795737;tmVar:rs3795737;VariantGroup:5;CorrespondingGene:81875;RS#:3795737

rs138355706;tmVar:rs138355706;VariantGroup:3;CorrespondingGene:6274;RS#:138355706

0no label

myc-pendrin @VARIANT$, L445W, Q446R, G672E were not co-immunoprecipitated with EphA2. Densitometric quantifications are shown (b). Mean +- SEM; one-way ANOVA with Bonferroni post hoc analyses; *p < 0.05; (n = 3). c, d Immunoprecipitation of EphA2 with mutated pendrin. Immunocomplex of myc-@GENE$ 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 S166N was not internalized after ephrin-B2 stimulation while @GENE$ and other mutated pendrins were not affected.

pendrin;20132

EphA2;20929

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

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

0no label

However, recently patients with defects in two components of this pathway and overlapping features of various forms of Noonan syndrome and @GENE$ and have been reported. 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 PTPN11 mutation (@VARIANT$; p.T468M) and a novel, potentially pathogenic missense @GENE$ variant (@VARIANT$; p.P340S) replacing a rigid nonpolar imino acid with a polar amino acid at a highly conserved position.

neurofibromatosis 1;226

SOS1;4117

c.1403 C > T;tmVar:c|SUB|C|1403|T;HGVS:c.1403C>T;VariantGroup:6;CorrespondingGene:5781;RS#:121918457;CA#:220134

c.1018 C > T;tmVar:c|SUB|C|1018|T;HGVS:c.1018C>T;VariantGroup:2;CorrespondingGene:6654;RS#:190222208;CA#:1624660

0no label

We report digenic variants in SCRIB and PTK7 associated with NTDs in addition to @GENE$ and CELSR1 heterozygous variants in additional NTD cases. The combinatorial variation of @GENE$ @VARIANT$ (p.P642R) 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.

SCRIB;44228

PTK7;43672

c.1925C > G;tmVar:c|SUB|C|1925|G;HGVS:c.1925C>G;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

Amino acid conservation analysis showed that seven of the 10 variants (CELSR1 p.G1122S, CELSR1 p.R769W, DVL3 @VARIANT$, @GENE$ p.P642R, SCRIB p.G1108E, @GENE$ @VARIANT$ and SCRIB p.K618R) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

PTK7;43672

SCRIB;44228

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

p.G644V;tmVar:p|SUB|G|644|V;HGVS:p.G644V;VariantGroup:9;CorrespondingGene:23513;RS#:201104891;CA#:187609256

0no label

This is a rare variant with a reported frequency of T = 0.0006/3 (1000 Genomes), T = 0.0005/6 (GO-@GENE$), and T = 0.0005/66 (TOPMED) in various databases. Neither the @VARIANT$ nor the @VARIANT$ variant has been reported as a mutation of a compound heterozygote in patients diagnosed with a myopathy secondary to mutations in either the @GENE$ or CAPN genes.

ESP;148713

DES;56469

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

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

0no label

The nucleotide sequence showed a @VARIANT$ (c.769G>C) of the coding sequence in exon 7 of @GENE$, which results in the substitution of Gly at residue 257 to Arg. Additionally, the nucleotide sequence showed a monoallelic @VARIANT$ (c.511C>T) of the coding sequence in exon 3 of @GENE$, which results in the substitution of Arg at residue 171 to Cys.

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

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 (235delC/N166S, 235delC/A194T and @VARIANT$/@VARIANT$).

GJB3;7338

GJB2;2975

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

Only 9 mutations previously reported as recurrent were detected in our series of patients (i.e. 11% of the mutations), specifically, c.1996C>T, c.223delG, c.1556G>A, c.494C>T, c.3719G>A and @VARIANT$ in @GENE$, c.238_239dupC in USH1C, and c.2299delG and @VARIANT$ in @GENE$. Therefore, in the process of designing any strategy for USH molecular diagnosis, taking into account the high prevalence of novel mutations appears to be of major importance.

MYO7A;219

USH2A;66151

c.5749G>T;tmVar:c|SUB|G|5749|T;HGVS:c.5749G>T;VariantGroup:155;CorrespondingGene:4647;RS#:780609120;CA#:224854968

c.10712C>T;tmVar:c|SUB|C|10712|T;HGVS:c.10712C>T;VariantGroup:83;CorrespondingGene:7399;RS#:202175091;CA#:262060

0no label

Subsequently, genetic testing for the LQT1, LQT2, LQT3, LQT5, and LQT6 genes identified a heterozygous c.3092_3096dup (@VARIANT$) mutation of the KCNH2 gene (LQT2) and a heterozygous c.170T > C (@VARIANT$) 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 @GENE$. Genetic screening revealed that both sons are not carrying the familial KCNH2 mutation.

LQT2;201

LQT6;71688

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

p.Ile57Thr;tmVar:p|SUB|I|57|T;HGVS:p.I57T;VariantGroup:0;CorrespondingGene:9992;RS#:794728493

0no label

The proband from Family 1 is consistent with the H1 haplotype based on the presence of homozygous genotypes for rs4935 and @VARIANT$ although this is not definitive because the rs10277 and rs1065154 polymorphisms were not covered. The haplotype of the proband from Family 2 could not be determined based on the available genotype data. For Family 3, sequencing data were available for four family members, and we manually reconstructed the haplotype assuming the minimal number of recombinations. The result indicated that Family 3's haplotype was consistent with either the H2 or the H5 haplotype described in the study by Lucas et al.. On the basis of these results, our three families have at least two different haplotypes associated with the SQSTM1 mutation, indicating that this unique phenotype is not a haplotype-specific effect, as well as demonstrating that these families are not remotely related to each other. Discussion We present the first detailed clinical and pathologic data from three unrelated families with predominant distal myopathy associated with a known pathologic variant in @GENE$ (p.Pro392Leu) and a variant in @GENE$ (@VARIANT$).

SQSTM1;31202

TIA1;20692

rs4797;tmVar:rs4797;VariantGroup:0;CorrespondingGene:8878;RS#:4797

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

0no label

On the other hand, two missense mutations of the @GENE$ gene were identified in two families, SLC26A4: c.1300G>A (p.434A>T), EPHA2: c.1063G>A (p.G355R) and @GENE$: @VARIANT$ (p.410T>M), EPHA2: c.1532C>T (@VARIANT$) (Fig. 6a, b).

EPHA2;20929

SLC26A4;20132

c.1229C>A;tmVar:c|SUB|C|1229|A;HGVS:c.1229C>A;VariantGroup:21;CorrespondingGene:5172

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

0no label

Surprisingly, we identified two missense mutations in the proband: NM_001257180.2, exon10, @VARIANT$, p.His596Arg in @GENE$ (Figure 1c) and NM_002609.4, exon3, c.317G>C, @VARIANT$, rs544478083 in @GENE$ (Figure 1d). Subsequently, we further detected the distribution of the two variants in this family and found that the proband's father carried the SLC20A2 mutation, the proband's mother and maternal grandfather carried the PDGFRB variant (Figure 1a).

SLC20A2;68531

PDGFRB;1960

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

p.Arg106Pro;tmVar:p|SUB|R|106|P;HGVS:p.R106P;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

11

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) c.317G>C (p.Arg106Pro) in @GENE$ were identified. The proband's father with the SLC20A2 c.1787A>G (@VARIANT$) mutation showed obvious brain calcification but was clinically asymptomatic. The proband's mother with the PDGFRB c.317G>C (@VARIANT$) 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

p.Arg106Pro;tmVar:p|SUB|R|106|P;HGVS:p.R106P;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

0no label

We finally found evidence of four potential novel candidate genes contributing to IHH: coiled-coil domain containing 88C (CCDC88C), cell adhesion associated, oncogene regulated (CDON), glutamate decarboxylase like 1 (GADL1), and sprouty related EVH1 domain containing 3 (@GENE$). The CCDC88C missense variant p. Arg1299Cys was heterozygous in case P05. CCDC88Cis a negative regulator of the Wnt signaling pathway, and bi-allelic mutations in CCDC88C were linked to midline brain malformation. Of note, the same variant @VARIANT$ was previously reported in a patient affected with pituitary stalk interruption syndrome (PSIS) with an etiologic overlap of IHH, who carried a mutationinan IHH-causative gene, @GENE$ (TACR3). Similarly, the CCDC88C-mutated case P05 in our study carried additional variants in DCC netrin 1 receptor (DCC)p. Gln91Arg, and FGFR1 @VARIANT$, implying that the deleterious variants in CCDC88C act together with other variants to cause IHH through a digenic/oligogenic model.

SPRED3;28061

tachykinin receptor 3;824

p. Arg1299Cys;tmVar:p|SUB|R|1299|C;HGVS:p.R1299C;VariantGroup:4;CorrespondingGene:440193;RS#:142539336;CA#:7309192

c.1664-2A>C;tmVar:c|SUB|A|1664-2|C;HGVS:c.1664-2A>C;VariantGroup:25;CorrespondingGene:2260

0no label

33 Family 22 presented a complex case with three pathogenic alleles in the parents, among which the @GENE$: @VARIANT$ (p.S1448F) variant was a known pathogenic variant for adult-onset manifestation, while the foetal PKD (22.1) was inferred to have been caused by the compound heterozygous variants @GENE$: c.1675C > T (p.R559W) and PKHD1: c.7942G > A (@VARIANT$), which were inherited from the mother and the father, respectively (Figure 3).

PKD1;250

PKHD1;16336

c.4343C > T;tmVar:c|SUB|C|4343|T;HGVS:c.4343C>T;VariantGroup:8;CorrespondingGene:5310;RS#:546332839;CA#:7832402

p.G2648S;tmVar:p|SUB|G|2648|S;HGVS:p.G2648S;VariantGroup:6;CorrespondingGene:5314;RS#:139555370;CA#:149529

0no label

Patient P0432 has a c.4030_4037delATGGCTGG (@VARIANT$) mutation in @GENE$ and a missense mutation in @GENE$ (@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.

USH2A;66151

CDH23;11142

p.M1344fsX42;tmVar:p|FS|M|1344||42;HGVS:p.M1344fsX42;VariantGroup:306;CorrespondingGene:26798

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

11

In this line, an increased side chain polarity associated with amino acid substitution @VARIANT$ could also interfere protein interactions involving the first PITX2 transcriptional inhibitory domain, leading to a functional alteration. Additional studies are required to evaluate these hypotheses. Interestingly, according to Ensembl Regulatory Build, FOXC2 variants @VARIANT$ (synonymous) and c.*38T>G (non coding 3' UTR) also mapped at a promoter, which overlapped with FOXC2 and @GENE$-@GENE$ genes.

FOXC2;21091

AS1;736

p.(A188T);tmVar:p|SUB|A|188|T;HGVS:p.A188T;VariantGroup:5;CorrespondingGene:5308;RS#:77144743;CA#:203139

p.S36S;tmVar:p|SUB|S|36|S;HGVS:p.S36S;VariantGroup:0;CorrespondingGene:103752587;RS#:138318843;CA#:8218260

0no label

For example, two variants in proband P15, p. Ala103Val in PROKR2 and p. Tyr503His in DDB1 and CUL4 associated factor 17 (DCAF17), were inherited from unaffected father, while @GENE$ @VARIANT$ variant was inherited from unaffected mother. Proband 17 inherited @GENE$ @VARIANT$ and CDON p. Val969Ile variants from his unaffected father and mother, respectively.

DMXL2;41022

CHD7;19067

p. Gln1626His;tmVar:p|SUB|Q|1626|H;HGVS:p.Q1626H;VariantGroup:10;CorrespondingGene:23312;RS#:754695396;CA#:7561930

p. Trp1994Gly;tmVar:p|SUB|W|1994|G;HGVS:p.W1994G;VariantGroup:14;CorrespondingGene:55636

0no label

Digenic inheritances of @GENE$/@GENE$ and GJB2/GJB3 (group II). (A) In addition to @VARIANT$ in GJB2, the de novo variant of MITF, @VARIANT$ was identified in SH107-225. (B) There was no GJB6 large deletion within the DFNB1 locus.

GJB2;2975

MITF;4892

c.235delC;tmVar:c|DEL|235|C;HGVS:c.235delC;VariantGroup:10;CorrespondingGene:2706;RS#:80338943

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

0no label

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

EDN3;88

MITF;4892

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

Variants in all known WS candidate genes (EDN3, EDNRB, MITF, PAX3, SOX10, SNAI2, and @GENE$) were searched and a novel rare heterozygous deletion mutation (@VARIANT$; p.Asn322fs) was identified in the @GENE$ gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; p.Arg203Cys) and TYRO3 (c.1037T>A; @VARIANT$) gene was identified in the exome data of both patients.

TYRO3;4585

MITF;4892

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

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

0no label

Since the @GENE$ c.1664-2A>C variant was evaluated as pathogenic according to the ACMG guideline, this family might be considered as a case of monogenic inheritance. However, proband P05 also carried a paternal variant (@GENE$ p. Gln91Arg) and a maternal variant (CCDC88C @VARIANT$). Considering the facts that the loss-of-function mutations in FGFR1 were identified to act in concert with other gene defects and the CCDC88C p. Arg1299Cys variant was reported in a PSIS patient with an IHH-causative gene in a digenic manner, the possibility of oligogenic inheritance in family 05 cannot be ruled out. Six families harbored only one variant of IHH genes, but none had sufficient evidence to be identified as monogenic models. Among these variants, one was frameshift variant, immunoglobulin superfamily member 10 (IGSF10) @VARIANT$, and the rest were missense variants.

FGFR1;69065

DCC;21081

p. Arg1299Cys;tmVar:p|SUB|R|1299|C;HGVS:p.R1299C;VariantGroup:4;CorrespondingGene:440193;RS#:142539336;CA#:7309192

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

0no label

Two different @GENE$ mutations (N166S and A194T) occurring in compound heterozygosity with the @VARIANT$ and 299delAT of @GENE$ were identified in three unrelated families (235delC/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

Three rare missense variants (R2034Q, L2118V, and @VARIANT$) of the SPG11 gene were found. The high detection rate of missense variants of this gene is probably due to the large size of the coding region; therefore, we suggest that these SPG11 variants are unlikely to be deleterious. Variants in the @GENE$ 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 (M392V,) and a novel variant (Q84H) were found in the UBQLN2 gene. The novel @VARIANT$ variant affects the N-terminal ubiquitin-like domain of the ubiquilin-2 protein, which is involved in binding to proteasome subunits. FUS variants have been mostly detected in familial ALS cases that are localized within the C-terminus of the @GENE$ protein.

SPG11;41614

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

To examine whether EphA2 is involved in dysfunction of pendrin caused by these amino acid substitutions, the effect of @GENE$ L117F, pendrin @VARIANT$, and pendrin @VARIANT$ mutations on @GENE$ interaction and internalization was examined.

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

Two affected (II-3 and III-9) individuals were selected for WES. +/+, wild-type; +/-, heterozygous for REEP4 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 @GENE$ missense variant A TOR2A nonsynonymous SNV (c.568C>T [NM_130459.3], @VARIANT$ [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).

BSP+;3644

TOR2A;25260

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

p.Arg190Cys;tmVar:p|SUB|R|190|C;HGVS:p.R190C;VariantGroup:12;CorrespondingGene:27433;RS#:376074923;CA#:5250615

0no label

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

VAPB;36163

TAF15;131088

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

This individual was also heterozygous for the common @GENE$ @VARIANT$ variant, and also carries a rare glycine decarboxylase (GLDC) c.2203G>T missense variant, possibly indicating a compromised FOCM in this patient. Interestingly, 2 unrelated patients harbor an identical extremely rare (gnomAD frequency 1/276 358) missense variant (@VARIANT$; p.Val2517Met) within the transmembrane receptor domain of the cadherin, EGF LAG seven-pass G-type receptor 1 (@GENE$) gene, which encodes a core protein of the PCP pathway (Figure 2E, Table S2 in Appendix S3).

MTHFR;4349

CELSR1;7665

c.677C>T;tmVar:c|SUB|C|677|T;HGVS:c.677C>T;VariantGroup:27;CorrespondingGene:4524;RS#:1801133;CA#:170990

c.7549G>A;tmVar:c|SUB|G|7549|A;HGVS:c.7549G>A;VariantGroup:14;CorrespondingGene:9620;RS#:1261513383

0no label

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 @GENE$ 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$, resulting in an asparagine into serine substitution in codon 166 (@VARIANT$) and for the @VARIANT$ of GJB2 (Fig. 1b, d).

GJB2;2975

GJB3;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

In patient AVM427, the de novo heterozygous missense variant @VARIANT$ (p.Asp1148Tyr) was identified in ZFYVE16 (table 1), which encodes an endosomal protein also known as endofin. ZFYVE16 is an SMAD anchor that facilitates @GENE$ phosphorylation, thus activating BMP signalling. In addition to Smad1-mediated BMP signalling, @GENE$ also interacts with Smad4 to mediate Smad2-Smad4 complex formation and facilitate TGF-beta signalling, indicating a regulatory role in BMP/TGF-beta signalling (figure 3). Other potential dominant genes with incomplete penetrance We also examined other inherited dominant pathogenic variants potentially involving LoF. Evidence of involvement in the pathogenesis of AVM was found in patient AVM312, who carried a paternally inherited heterozygous nonsense variant, c.1891G>T (p.Glu631Ter), in EGFR (table 1). Oncogenic EGFR stimulates angiogenesis via the VEGF pathway. As a truncated germline EGFR variant has not been reported in humans, c.1891G>T (@VARIANT$) in patient AVM312 was classified as likely pathogenic and EGFR as a candidate gene due to the vital role of EGFR in EGF and VEGF signalling.

SMAD1;21196

ZFYVE16;8826

c.3442G>T;tmVar:c|SUB|G|3442|T;HGVS:c.3442G>T;VariantGroup:3;CorrespondingGene:9765

p.Glu631Ter;tmVar:p|SUB|E|631|X;HGVS:p.E631X;VariantGroup:8;RS#:909905659

0no label

Two SALS patients carried multiple ALS-associated variants that are rare in population databases (ANG p.K41I with VAPB p.M170I and @GENE$ @VARIANT$ with SETX p.I2547T and @GENE$ @VARIANT$).

TAF15;131088

SETX;41003

p.R408C;tmVar:p|SUB|R|408|C;HGVS:p.R408C;VariantGroup:9;CorrespondingGene:8148;RS#:200175347;CA#:290041127

p.T14I;tmVar:p|SUB|T|14|I;HGVS:p.T14I;VariantGroup:28;CorrespondingGene:4094;RS#:1219381953

11

The ages of onset of the patients with the @GENE$ 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, @VARIANT$) were detected. The P11S variant affects the b isoform of the @GENE$ 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 DYNC1H1 gene.

ALS2;23264

MATR3;7830

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

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

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; @VARIANT$); a pathogenic new homozygous nucleotide change in @GENE$ that leads to a stop codon in position 255, c.763A > T, and a likely pathogenic homozygous substitution c.1235G > T in BBS6, leading to the change p.(@VARIANT$).

BBS2;12122

BBS7;12395

p.(Asn354Lys);tmVar:p|SUB|N|354|K;HGVS:p.N354K;VariantGroup:23;CorrespondingGene:583

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

0no label

Variants in all known WS candidate genes (EDN3, @GENE$, MITF, PAX3, SOX10, SNAI2, and @GENE$) were searched and a novel rare heterozygous deletion mutation (@VARIANT$; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (@VARIANT$; p.Arg203Cys) and TYRO3 (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients.

EDNRB;89

TYRO3;4585

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

As shown in Table 3, all male individuals carrying the @GENE$-Q1916R mutation with (II-4, III-1, III-5 and IV-3) or without (III-7) concomitant SCN5A-R1193Q showed the ERS phenotypes. The female CACNA1C-@VARIANT$ mutation carriers with @GENE$-@VARIANT$ variant (II-3, II-6, III-4 and IV-1) were not affected, while the female member only carrying the CACNA1C-Q1916R mutation (IV-4) showed the ER ECG pattern.

CACNA1C;55484

SCN5A;22738

Q1916R;tmVar:p|SUB|Q|1916|R;HGVS:p.Q1916R;VariantGroup:4;CorrespondingGene:775;RS#:186867242;CA#:6389963

R1193Q;tmVar:p|SUB|R|1193|Q;HGVS:p.R1193Q;VariantGroup:7;CorrespondingGene:6331;RS#:41261344;CA#:17287

0no label

Compared to WT (wild-type) proteins, we found that the ability of GFP-CYP1B1 @VARIANT$ and GFP-@GENE$ E229K to immunoprecipitate HA-TEK @VARIANT$ and HA-@GENE$ Q214P, respectively, was significantly diminished.

CYP1B1;68035

TEK;397

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

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

0no label

The nucleotide sequence showed a G to C transition at nucleotide 769 (@VARIANT$) of the coding sequence in exon 7 of EDA, which results in the substitution of Gly at residue 257 to Arg. Additionally, the nucleotide sequence showed a monoallelic C to T transition at nucleotide 511 (c.511C>T) of the coding sequence in exon 3 of WNT10A, which results in the substitution of Arg at residue 171 to Cys. DNA sequencing of the parents' genome revealed that both mutant alleles were from their mother (Fig. 2A), who carried a heterozygous EDA mutation (c.769G>C) and a heterozygous WNT10A c.511C>T mutation, and showed absence of only the left upper lateral incisor without other clinical abnormalities. No mutations in these genes were found in the father. Sequence analyses of EDA and WNT10A genes. (A) The @GENE$ mutation c.769G>C and @GENE$ mutation @VARIANT$ were found in patient N1, who 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

Given the reported normal function of pendrin L117F and @GENE$ 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 pendrin caused by these amino acid substitutions, the effect of pendrin L117F, pendrin @VARIANT$, and pendrin F355L mutations on EphA2 interaction and internalization was examined. While the amount of co-precipitated pendrin mutants with EphA2 was comparable to that of wild type (wt) pendrin (Fig. 5c, d), the S166N mutant failed to be internalized after ephrin-B2 stimulation (Fig. 5e, f). Taken together, these results further demonstrate that @GENE$ 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.

pendrin;20132

EphA2;20929

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

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

0no label

Moller et al. reported an index case with digenic variants in @GENE$ (@VARIANT$) and MYBPC3 (R326Q), both encoding sarcomeric proteins that are likely to affect its structure when mutated. Petropoulou et al. reported a family severely affected by DCM and who had two digenic variations in MYH7 (Asp955Asn) and @GENE$ (@VARIANT$), both sarcomeric genes.

MYH7;68044

TNNT2;68050

L1038P;tmVar:p|SUB|L|1038|P;HGVS:p.L1038P;VariantGroup:8;CorrespondingGene:4625;RS#:551897533;CA#:257817954

Asn83His;tmVar:p|SUB|N|83|H;HGVS:p.N83H;VariantGroup:4;CorrespondingGene:7139;RS#:1060500235

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 @VARIANT$ 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 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

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

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

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 p.Arg153Cys (@VARIANT$) mutation was found in exon 3 of EDA, it results in the substitution of Arg at residue 153 to Cys. Moreover, a heterozygous @VARIANT$ (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

c.457C>T;tmVar:c|SUB|C|457|T;HGVS:c.457C>T;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 @VARIANT$ (p.His596Arg) 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 c.317G>C (@VARIANT$) 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

p.Arg106Pro;tmVar:p|SUB|R|106|P;HGVS:p.R106P;VariantGroup:1;CorrespondingGene:5159;RS#:544478083

0no label

@GENE$ mutations were the second most prevalent genetic alterations in DH: five different heterozygous variants were found in 5/21 patients (23.8%), and these often cooccurred with DUOX2 or DUOXA2 mutations. DUOX2 and TG mutation locations varied in the corresponding proteins (Figure 2). Additionally, three DUOXA2 variants were found in 3/21 patients (14%), and a known heterozygous variant in SLC26A4 was found in one patient. No mutations in SLC5A5, TPO, or IYD gene exons were found. Most of the variants presented as heterozygous in patients. Only three variants were homozygous in three patients: (1) DUOX2: @VARIANT$ (p.M927V) in one patient, (2) @GENE$:c.3329G>A (p.R1110Q) in one patient, and (3) DUOXA2: @VARIANT$ (p.Y138X) in one patient.

TG;2430

DUOX2;9689

c.2779A>G;tmVar:c|SUB|A|2779|G;HGVS:c.2779A>G;VariantGroup:27;CorrespondingGene:50506;RS#:755186335;CA#:7538155

c.413dupA;tmVar:c|DUP|413|A|;HGVS:c.413dupA;VariantGroup:19;CorrespondingGene:405753;RS#:1085307064

0no label

WES revealed heterozygous mutations in two genes known to affect hypothalamic and pituitary development: c.253C>T;@VARIANT$ in @GENE$ (MIM 607123; NM_144773.2; rs141090506) inherited from an unaffected mother and c.1306A>G;p.I436V in @GENE$ (MIM 606417; NM_018117.11; @VARIANT$) inherited from an unaffected father, both confirmed by Sanger sequencing (Fig. 1).

PROKR2;16368

WDR11;41229

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

rs34602786;tmVar:rs34602786;VariantGroup:3;CorrespondingGene:55717;RS#:34602786

11

To investigate the role of @GENE$ 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 @GENE$ in 3 simplex families (235delC/@VARIANT$, 235delC/A194T and 299delAT/A194T).

GJB3;7338

GJB2;2975

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

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

0no label

Both mutations are novel and whilst a different mutation, R80W, has been reported in @GENE$, further evidence to support the pathogenicity of @VARIANT$ is lacking. The siblings we describe with the @GENE$ P291fsinsC and HNF4A @VARIANT$ mutations are the first cases of digenic transcription factor MODY where both mutations have previously been reported as being pathogenic.

HNF4A;395

HNF1A;459

E508K;tmVar:p|SUB|E|508|K;HGVS:p.E508K;VariantGroup:0;CorrespondingGene:6927;RS#:483353044;CA#:289173

R127W;tmVar:p|SUB|R|127|W;HGVS:p.R127W;VariantGroup:3;CorrespondingGene:3172;RS#:370239205;CA#:9870226

0no label

Using these filtering settings, no variants in @GENE$ or @GENE$ were detected in any of our control datasets which emphasizes that the presence of rare double hits in our FTLD-TDP cohort is unlikely to have occurred by chance alone. Comparison of sequence traces of OPTN exon 8 (harboring the @VARIANT$ mutation) in gDNA and mRNA prepared from cerebellar cortex of case A showed the absence of mutant RNA (T-allele) suggesting the degradation of mutant RNA by nonsense mediated decay (Figure 1c). A similar analysis of OPTN exon 14 (harboring the @VARIANT$) mutation showed significantly reduced amounts of the wild-type (C-allele) in the cDNA sequence suggesting that the missense variant was inherited in trans with respect to the OPTN nonsense mutation (Figure 1c).

OPTN;11085

TBK1;22742

p.Gln235*;tmVar:p|SUB|Q|235|*;HGVS:p.Q235*;VariantGroup:26;CorrespondingGene:29110

p.Ala481Val;tmVar:p|SUB|A|481|V;HGVS:p.A481V;VariantGroup:1;CorrespondingGene:10133;RS#:377219791;CA#:5410970

0no label

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

PDGFRB;1960

SLC20A2;68531

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

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

0no label