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

WNT10A;22525

EDA;1896

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

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

0no label

Deleterious variants in HS1BP3 (NM_022460.3: @VARIANT$, p.Gly32Cys) and GNA14 (NM_004297.3: @VARIANT$, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP. Deleterious variants in @GENE$,@GENE$,CAPN11,VPS13C,UNC13B,SPTBN4,MYOD1, and MRPL15 were found in two or more independent pedigrees.

DNAH17;72102

TRPV4;11003

c.94C>A;tmVar:c|SUB|C|94|A;HGVS:c.94C>A;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

C2orf74 gene might interact with @GENE$ gene product and give rise to the spectrum of phenotype varying from severe phenotype with complete penetrance to partial features. Conclusion In this study, we analysed a large family segregating Waardenburg syndrome type 2 to identify the underlying genetic defects. Whole genome SNP genotyping, whole exome sequencing and segregation analysis using Sanger approach was performed and a novel single nucleotide deletion mutation (@VARIANT$) in the MITF gene and a rare heterozygous, missense damaging variant (@VARIANT$; p.Val34Gly) in the @GENE$ was identified.

MITF;4892

C2orf74;49849

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

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

0no label

Patient P0418 carries a nonsense mutation in USH2A (@VARIANT$) and a missense mutation in MYO7A (p.K268R), but his brother, who is also clinically affected, does not carry the MYO7A mutation. Patient P0432 has a c.4030_4037delATGGCTGG (p.M1344fsX42) mutation in USH2A and a missense mutation in @GENE$ (p.R1189W), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (c.46C>G; @VARIANT$) and USH2A (c.9921T>G). Her father carries the mutations in MYO7A and USH2A without displaying symptoms of the disease, whilst her unaffected mother carries the mutation in USH1G. The mutations in MYO7A, USH1G and @GENE$ were not found in 666 control alleles.

CDH23;11142

USH2A;66151

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

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

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 (c.965delA; @VARIANT$) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (@VARIANT$; p.Arg203Cys) and TYRO3 (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients.

EDNRB;89

TYRO3;4585

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

The p.Ile312Met (c.936C>G) mutation in EDA and heterozygous p.Arg171Cys (@VARIANT$) mutation in WNT10A were detected. The coding sequence in exon 9 of @GENE$ showed a C to G transition, which results in the substitution of @VARIANT$; also, the coding sequence in exon 3 of @GENE$ showed a C to T transition at nucleotide 511, which results in the substitution of Arg at residue 171 to Cys.

EDA;1896

WNT10A;22525

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

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

0no label

25 The RYR3 (NM_001036: c.7812C > G, @VARIANT$) and EBNA1BP2 (NM_001159936: c.1034A > T, p.Asn345Ile) variants were classified as likely benign and benign, respectively, while the TRIP6 (NM_003302: @VARIANT$, p.Glu274Asp) and the @GENE$ (NM_006615: c.55G > T, p.Ala19Ser) variants were classified as VUS. 21 TRIP6 promotes cell migration and invasion through Wnt/beta-catenin signaling and was shown to be upregulated in colorectal tumors. 24 Therefore, TRIP6 variants that increase protein stability or expression could potentially stimulate colorectal tumorigenesis. In addition, lost-of-function variants in CAPN9 might promote tumor formation, as Calpain-9 induces cell cycle arrest and apoptosis, and low expression predicts a poorer prognosis in gastric cancer patients. 25 The contribution of the genetic variants, other than @GENE$ and MUTYH, to cancer risk cannot be completely excluded.

CAPN9;38208

MSH6;149

p.Asn2604Lys;tmVar:p|SUB|N|2604|K;HGVS:p.N2604K;VariantGroup:10;CorrespondingGene:6263;RS#:41279214;CA#:7459988

c.822G > C;tmVar:c|SUB|G|822|C;HGVS:c.822G>C;VariantGroup:22;CorrespondingGene:7205;RS#:76826261;CA#:4394675

0no label

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 DES or CAPN genes. Discussion The patient's history, clinical examination, EMG testing, muscle biopsy results, and the lack of response to any therapy suggest that he does not have an inflammatory myopathy but rather a genetic disorder. Mutations in CAPN3 and DES genes result in LGMD inherited in an autosomal recessive pattern. Homozygous or compound heterozygous mutations in the @GENE$ and DES genes cause LGMD 2A and @GENE$, respectively.

CAPN3;52

LGMD 2R;56469

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

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

0no label

Below symbols are indicated genotypes for @GENE$ and PITX2, age at diagnosis and number or surgical operations per eye, respectively. M1, CYP1B1: p.(A179fs*18). M2, CYP1B1: @VARIANT$. M3, CYP1B1: p.(E173*). M4, PITX2: p.(P179T). M5, PITX2: @VARIANT$. Arrows show the index cases. +: wild-type allele. The asterisk indicates a de novo PITX2 variant. Evolutionary conservation of FOXC2 and @GENE$ variants Evolutionary amino acid or nucleotide sequence conservation analysis were assessed using a multiple sequence alignment of representative orthologous proteins or genes of seven different species, from fish to human.

CYP1B1;68035

PITX2;55454

p.(E387K);tmVar:p|SUB|E|387|K;HGVS:p.E387K;VariantGroup:2;CorrespondingGene:1545;RS#:55989760;CA#:254241

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

0no label

For example, @GENE$:@VARIANT$, which occurred in 0/384 control chromosomes and was predicted to be "probably damaging" by the Polyphen program, was found with a homozygous @GENE$ nonsense mutation (@VARIANT$) (Case #15).

USH1C;77476

CDH23;11142

p.Tyr813Asp;tmVar:p|SUB|Y|813|D;HGVS:p.Y813D;VariantGroup:3;CorrespondingGene:10083

p.Arg2107X;tmVar:p|SUB|R|2107|X;HGVS:p.R2107X;VariantGroup:4;CorrespondingGene:64072;RS#:773945008

11

Results Cosegregating deleterious variants (GRCH37/hg19) in @GENE$ (NM_001127222.1: c.7261_7262delinsGT, p.Pro2421Val), REEP4 (NM_025232.3: c.109C>T, @VARIANT$), TOR2A (NM_130459.3: c.568C>T, p.Arg190Cys), and ATP2A3 (NM_005173.3: c.1966C>T, p.Arg656Cys) were identified in four independent multigenerational pedigrees. Deleterious variants in HS1BP3 (NM_022460.3: @VARIANT$, p.Gly32Cys) and @GENE$ (NM_004297.3: c.989_990del, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP.

CACNA1A;56383

GNA14;68386

p.Arg37Trp;tmVar:p|SUB|R|37|W;HGVS:p.R37W;VariantGroup:10;CorrespondingGene:80346;RS#:780399718;CA#:4663211

c.94C>A;tmVar:c|SUB|C|94|A;HGVS:c.94C>A;VariantGroup:25;CorrespondingGene:64342

0no label

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$ (@VARIANT$) and a variant in @GENE$ (@VARIANT$).

SQSTM1;31202

TIA1;20692

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

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

11

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

LQT5;71688

LQT2;201

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

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

0no label

The @VARIANT$ (c.936C>G) mutation in EDA and heterozygous p.Arg171Cys (c.511C>T) mutation in @GENE$ were detected. The coding sequence in exon 9 of @GENE$ showed a C to G transition, which results in the substitution of Ile at residue 312 to Met; also, the coding sequence in exon 3 of WNT10A showed a @VARIANT$, which results in the substitution of Arg at residue 171 to Cys.

WNT10A;22525

EDA;1896

p.Ile312Met;tmVar:p|SUB|I|312|M;HGVS:p.I312M;VariantGroup:7;CorrespondingGene:1896

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

In the individual carrying the P505L NEFH variant, an additional novel alteration (@VARIANT$) was detected in the GRN gene. Loss-of-function GRN variants are primarily considered to cause frontotemporal lobar degeneration, but there is evidence that missense @GENE$ 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 @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 @GENE$ gene were originally reported in Paget's disease of bone.

GRN;1577

SQSTM1;31202

C335R;tmVar:p|SUB|C|335|R;HGVS:p.C335R;VariantGroup:29;CorrespondingGene:29110;RS#:1383907519

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

0no label

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 @VARIANT$ was not internalized after @GENE$ stimulation while EphA2 and other mutated pendrins were not affected.

pendrin;20132

ephrin-B2;3019

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

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

0no label

We observed that in 5 PCG cases heterozygous @GENE$ mutations (p.A115P, p.E229 K, and @VARIANT$) co-occurred with heterozygous TEK mutations (p.E103D, p.I148T, p.Q214P, and p.G743A) indicating a potential digenic inheritance (Fig. 1a). None of the normal controls carried both the heterozygous combinations of CYP1B1 and @GENE$ mutations. The TEK Q214P and G743A alleles were absent in 1024 controls, whereas very low frequencies of heterozygous TEK @VARIANT$ (0.005) and I148T (0.016) alleles were found in the control population (Table 1).

CYP1B1;68035

TEK;397

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

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

0no label

@GENE$E Molecular genetics of primary congenital glaucoma Digenic inheritance in medical genetics Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21 The trabecular meshwork outflow pathways: structural and functional aspects A lymphatic defect causes ocular hypertension and glaucoma in mice Role of CYP1B1 in glaucoma Digenic inheritance of early-onset glaucoma: CYP1B1, a potential modifier gene Common and rare genetic risk factors for glaucoma Genetic interaction of TEK and CYP1B1 in PCG patients. a Pedigrees of four PCG families segregating the heterozygous TEK and CYP1B1 alleles. The affected probands are indicated by solid black symbols who harbor both the heterozygous mutant alleles, while their asymptomatic parents carry either of the corresponding heterozygous TEK or @GENE$ alleles. The genotypes of the individuals for the TEK and CYP1B1 mutations are indicated below the symbols. Asterisk indicates the siblings whose biological samples were unavailable. Co-segregation of TEK @VARIANT$ and CYP1B1 @VARIANT$ was observed in two pedigrees and only a representative pedigree is shown.

INPP5;8682

CYP1B1;68035

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

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

0no label

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

GJB3;7338

Cx26;2975

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

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

0no label

Only three variants were homozygous in three patients: (1) DUOX2: c.2779A>G (p.M927V) in one patient, (2) @GENE$:c.3329G>A (@VARIANT$) in one patient, and (3) @GENE$: @VARIANT$ (p.Y138X) in one patient.

DUOX2;9689

DUOXA2;57037

p.R1110Q;tmVar:p|SUB|R|1110|Q;HGVS:p.R1110Q;VariantGroup:22;CorrespondingGene:50506;RS#:368488511;CA#:7537915

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

0no label

A single @GENE$ mutation (@VARIANT$) has been linked to MRV in one family and an unrelated patient. This patient was subsequently found to carry a coexisting @GENE$ variant (c.1070A>G, @VARIANT$) by Evila et al.. Evila et al.'s study reported also an additional sporadic MRV case carrying the same TIA1 variant but a different 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)

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

11

Three patients carried missense variants both in FZD 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; @VARIANT$), and 465F99 (rare @GENE$ missense variant c.211C>T and a novel @GENE$ missense variant @VARIANT$).

FZD1;20750

FAT4;14377

c.10384A>G;tmVar:c|SUB|A|10384|G;HGVS:c.10384A>G;VariantGroup:2;CorrespondingGene:4824;RS#:373263457;CA#:4677776

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

0no label

In patient AVM359, one heterozygous VUS (@VARIANT$ [@VARIANT$]) in @GENE$ inherited from the mother and one likely pathogenic de novo heterozygous variant (c.1592G>A [p.Cys531Tyr]) in SCUBE2 were identified (online supplementary table S2). SCUBE2 functions as a coreceptor that enhances VEGF/@GENE$ binding to stimulate VEGF signalling.

ENG;92

VEGFR2;55639

c.589C>T;tmVar:c|SUB|C|589|T;HGVS:c.589C>T;VariantGroup:2;CorrespondingGene:83394;RS#:2229778;CA#:2061380

p.Arg197Trp;tmVar:p|SUB|R|197|W;HGVS:p.R197W;VariantGroup:2;CorrespondingGene:2022;RS#:2229778

0no label

Both sisters inherited the HNF4A gene mutation @VARIANT$ from their mother and the @GENE$ gene mutation P291fsinsC (@VARIANT$) from their father. The father was diagnosed with diabetes at 45 years of age. Their brother is heterozygous for the @GENE$ R127W mutation.

HNF1A;459

HNF4A;395

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

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

0no label

Similarly, the CCDC88C-mutated case P05 in our study carried additional variants in DCC netrin 1 receptor (@GENE$)@VARIANT$, and FGFR1 @VARIANT$, implying that the deleterious variants in @GENE$ act together with other variants to cause IHH through a digenic/oligogenic model.

DCC;21081

CCDC88C;18903

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

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

0no label

CONCLUSIONS We firstly identified the novel digenic heterozygous mutations by WES, KCNH2 p.307_308del and @GENE$ @VARIANT$, which resulted in LQTS with repeat syncope, torsades de pointes, ventricular fibrillation, and sinoatrial node dysfunction. @GENE$ @VARIANT$ may affect the function of Kv11.1 channel in cardiomyocytes by inducing a regional double helix of the amino acids misfolded and largest hydrophobic domain disorganized.

SCN5A;22738

KCNH2;201

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

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

0no label

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

EDN3;88

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

Moreover, patients carrying a @GENE$ Pro943Leu mutation have a significantly reduced extracellular matrix (ECM) in cardiomyocytes. These findings support the importance of LAMA4 as a structural and signalling molecule in cardiomyocytes, and may indicate the modifier role that missense variations in LAMA4 play in the disease. Digenic heterozygosity has been described in some DCM cases and is often associated with a severe presentation of DCM. Moller et al. reported an index case with digenic variants in MYH7 (@VARIANT$) and @GENE$ (@VARIANT$), both encoding sarcomeric proteins that are likely to affect its structure when mutated.

LAMA4;37604

MYBPC3;215

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

R326Q;tmVar:p|SUB|R|326|Q;HGVS:p.R326Q;VariantGroup:6;CorrespondingGene:4607;RS#:34580776;CA#:16212

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: @GENE$/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; p.Cys163del of @GENE$) and NELF/TACR3 (c. 1160-13C>T of NELF and @VARIANT$; @VARIANT$ of TACR3).

NELF;10648

KAL1;55445

c.824G>A;tmVar:c|SUB|G|824|A;HGVS:c.824G>A;VariantGroup:1;CorrespondingGene:26012;RS#:144292455;CA#:144871

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

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, @VARIANT$, p.R268C (two patients), @VARIANT$, 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.Y217D;tmVar:p|SUB|Y|217|D;HGVS:p.Y217D;VariantGroup:13;CorrespondingGene:3730

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

0no label

(b) The changed site of SCN5A gene (position 1864) increased the corresponding amino acid residues and nearby sequences hydrophobicity, but the influence was not significant DISCUSSION In our study, the proband with overlapped phenotypes of young early-onset LQTS and sinoatrial node dysfunction was first demonstrated to carry with the digenic heterozygous and pathogenic mutations of @GENE$ @VARIANT$ and @GENE$ @VARIANT$. KCNH2 p.307_308del induced a regional double helix of the amino acids misfolded and largest hydrophobic domain disorganized, which thus mainly caused LQTS.

KCNH2;201

SCN5A;22738

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

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

11

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 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

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

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

0no label

Bioinformatic analysis predicted that @GENE$-G38S was "tolerated" and @GENE$-C108Y was "damaging", whereas divergent results were obtained for KCNQ1-R583H and KCNH2-K897T, i.e., some programs considered these variants "damaging" and others as "benign" (Table 2). Moreover, the MAF of KCNQ1-p.@VARIANT$ was much smaller (0.000016) than the estimated prevalence of LQTS (0.0005), whereas the MAFs of KCNH2-p.K897T and KCNE1-@VARIANT$ were much larger (0.187 and 0.352, respectively).

KCNE1;3753

KCNH2;201

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

The affected probands are indicated by solid black symbols who harbor both the heterozygous mutant alleles, while their asymptomatic parents carry either of the corresponding heterozygous @GENE$ or CYP1B1 alleles. The genotypes of the individuals for the TEK and CYP1B1 mutations are indicated below the symbols. Asterisk indicates the siblings whose biological samples were unavailable. Co-segregation of TEK p.I148T and CYP1B1 @VARIANT$ was observed in two pedigrees and only a representative pedigree is shown. b Chromatograms of the four probands (lower panel) harboring the four different heterozygous TEK mutations. The site of nucleotide change is indicated by an arrow, compared to the corresponding wild-type sequence (upper panel). c TEK protein sequence conservation across different species for the four mutations (p.E103D, @VARIANT$, p.Q214P, and p.G743A). The conserved residue for each mutation is highlighted in blue color. d Schematic representation of the TEK and CYP1B1 domains (Ig immunoglobulin, EGF epidermal growth factor, FN fibronectin, TM transmembrane, M membrane, H hinge region) indicating the location of the mutations identified in PCG (color figure online) TEK and @GENE$ interact in cells.

TEK;397

CYP1B1;68035

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

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

0no label

Moreover, a heterozygous p.Gly213Ser (c.637G>A) mutation was detected in exon 3 of @GENE$, this leads to the substitution of Gly at residue 213 to Ser. Sequence analyses revealed that both mutant alleles were from his mother (Fig. 2D), who had a very mild phenotype of isolated tooth agenesis. His father did not have mutations in either of these genes. "S3" is a 14-year-old girl who had the typical clinical characteristics of HED: sparse hair, 26 missing permanent teeth, hypohidrosis, dry skin, and eczema on her body, but no plantar hyperkeratosis or nail abnormalities (Table 1). The heterozygous @VARIANT$ (c.466C>T) mutation was found in exon 3 of @GENE$, it results in the substitution of Arg at residue 156 to Cys. Additionally, the monoallelic @VARIANT$ (c.637G>A) mutation was also detected in exon 3 of WNT10A, it results in the substitution of Gly at residue 213 to Ser.

WNT10A;22525

EDA;1896

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

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

0no label

We identified four genetic variants (KCNQ1-@VARIANT$, @GENE$-p.C108Y, KCNH2-p.K897T, and @GENE$-@VARIANT$) in an LQTS family.

KCNH2;201

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

None of 2,504 self-declared healthy individuals in TGP has both @GENE$, c.1070A > G (@VARIANT$) and @GENE$, c.1175C > T (p.Pro392Leu). No other pathogenic or suspected pathogenic variants in genes associated with muscle diseases were identified in the proband of family 2 by expanded NGS panel studies or in the proband of family 1 by WES analysis. We are aware of a prior study in which this SQSTM1 mutation may be part of a common founder haplotype including the following four loci: [Chr5: 179260153C/T, refSNP ID rs4935; Chr5: @VARIANT$, rs4797; Chr5: 179264731T/C, rs10277; Ch5: 179264915G/T, rs1065154 ].

TIA1;20692

SQSTM1;31202

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

179260213G/A;tmVar:c|SUB|G|179260213|A;HGVS:c.179260213G>A;VariantGroup:0;CorrespondingGene:8878;RS#:4797;CA#:3600734

0no label

Interestingly, we found just one patient with variants in @GENE$, the most frequently detected gene in BBS patients. We identified a novel variant in BBS1 patient #10 c.1285dup (p.(Arg429Profs*72)) defined as pathogenic that segregates with phenotype together with c.46A > T (@VARIANT$, defined as likely pathogenic. A new pathogenic variant in BBS2 affecting a conserved residue in the functional domain of BBsome protein (c.1062C > G; @VARIANT$) was found in compound heterozygous state in patient #1 together with the known pathogenic variant p.(Arg339*). A new homozygous nucleotide change in @GENE$ that leads to a stop codon in position 255, c.763A > T, was identified in patient #3.

BBS1;11641

BBS7;12395

p.(Ser16Cys);tmVar:p|SUB|S|16|C;HGVS:p.S16C;VariantGroup:5;CorrespondingGene:582;RS#:772917364

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

0no label

myc-pendrin A372V, @VARIANT$, Q446R, G672E were not co-immunoprecipitated with EphA2. Densitometric quantifications are shown (b). Mean +- SEM; one-way ANOVA with Bonferroni post hoc analyses; *p < 0.05; (n = 3). c, d Immunoprecipitation of EphA2 with mutated pendrin. Immunocomplex of myc-pendrin L117F, @VARIANT$ and F355L was not affected. Densitometric quantifications are shown (d). Mean +- SEM; (n = 3). e, f Internalization of @GENE$ and mutated pendrin triggered by ephrin-B2 stimulation. Pendrin S166N was not internalized after @GENE$ stimulation while EphA2 and other mutated pendrins were not affected.

EphA2;20929

ephrin-B2;3019

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

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

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 c.730C>G), 2F07 (@GENE$ c.8807C>T and @GENE$ c.1622C>T), 618F05 (CELSR1 @VARIANT$ and SCRIB @VARIANT$).

CELSR1;7665

DVL3;20928

c.8282C>T;tmVar:c|SUB|C|8282|T;HGVS:c.8282C>T;VariantGroup:4;CorrespondingGene:9620;RS#:144039991;CA#:10292903

c.3979G>A;tmVar:c|SUB|G|3979|A;HGVS:c.3979G>A;VariantGroup:31;CorrespondingGene:23513;RS#:201563528;CA#:4918429

0no label

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 @VARIANT$. Additionally, the monoallelic p.Gly213Ser (c.637G>A) mutation was also detected in exon 3 of WNT10A, it results in the substitution of Gly at residue 213 to Ser.

WNT10A;22525

EDA;1896

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

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

0no label

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 MITF gene in both patients. Moreover, heterozygous missense variants in @GENE$ (c.607C>T; p.Arg203Cys) and TYRO3 (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

TYRO3;4585

SNAI3;8500

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

Amino acid conservation analysis showed that seven of the 10 variants (CELSR1 p.G1122S, CELSR1 p.R769W, DVL3 p.R148Q, @GENE$ @VARIANT$, @GENE$ @VARIANT$, SCRIB p.G644V and SCRIB p.K618R) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

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

Digenic inheritances of GJB2/@GENE$ and @GENE$/GJB3 (group II). (A) In addition to @VARIANT$ 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.

MITF;4892

GJB2;2975

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

Molecular Data All three probands carry two heterozygous variants: SQSTM1, c.1175C>T (p.Pro392Leu), and TIA1, @VARIANT$ (p.Asn357Ser). None of the unaffected family members harbor both variants (Figure 1). The @GENE$ variant and SQSTM1 variants have been reported in multiple databases. The @GENE$ variant is designated as @VARIANT$ in dbSNP and reported at allele frequencies of 0.0009 in the Exome Aggregation Consortium database (ExAC), 0.0024 in 1,000 Genomes Project database (TGP), and 0.0015 in the NHLBI GO Exome Sequencing Project (GO-ESP) (accessed January 23, 2018) and is more frequent in certain European populations.

TIA1;20692

SQSTM1;31202

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

rs104893941;tmVar:rs104893941;VariantGroup:1;CorrespondingGene:8878;RS#:104893941

0no label

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

SMAD1;21196

BMP;55955

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

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

0no label

(C) The sequence of the @VARIANT$ variant is well-conserved from humans to tunicates. (D) SH175-389 harbored a monoallelic @VARIANT$ variant of GJB2 and a monoallelic p.A194T variant of GJB3. DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = gap junction protein beta 2, GJB3 = @GENE$, GJB6 = @GENE$, MITF = microphthalmia-associated transcription factor.

gap junction protein beta 3;7338

gap junction protein beta 6;4936

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

p.V193E;tmVar:p|SUB|V|193|E;HGVS:p.V193E;VariantGroup:21;CorrespondingGene:2706

0no label

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

KCNH2;201

KCNQ1;85014

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

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

0no label

By contrast, the expression of human @GENE$ and @GENE$, either alone or in combination, did not restore the viability of the mutant (Fig 3C), suggesting that the human orthologs have evolved in structure and function in comparison to Gcn5. As the mutated amino acid in KAT2B, F307, is conserved in Drosophila Gcn5 (corresponding to Gcn5 F304), we re-expressed Gcn5 F304S in the Gcn5E333st hemizygous background (Gcn5 @VARIANT$). As a negative control, we re-expressed a predicted potentially damaging KAT2B variant (S502F corresponding to Gcn5 @VARIANT$) found in a homozygous state in a healthy individual from our in-house database.

KAT2A;41343

KAT2B;20834

F304S;tmVar:p|SUB|F|304|S;HGVS:p.F304S;VariantGroup:6;CorrespondingGene:39431

S478F;tmVar:p|SUB|S|478|F;HGVS:p.S478F;VariantGroup:13;CorrespondingGene:2648

0no label

Recurrent Variants Identified in Our Regressive Autism Cohort In our sequenced cohort of 134 individuals with autism and regression, we identified two recurrent variants, @GENE$ c.28C > A (@VARIANT$) and @GENE$ c.742C > T (@VARIANT$).

GRIN2A;645

PLXNB2;66630

p.Leu10Met;tmVar:p|SUB|L|10|M;HGVS:p.L10M;VariantGroup:0;CorrespondingGene:2903

p.Arg248Cys;tmVar:p|SUB|R|248|C;HGVS:p.R248C;VariantGroup:9;CorrespondingGene:23654;RS#:779647430;CA#:10313520

0no label

Additionally, a monoallelic @VARIANT$ (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 @GENE$ allele was from his mother (Fig. 2C), however, we were unable to screen for WNT10A mutations because of insufficient DNA. "S2" is a 17-year-old boy who had curly hair, 17 missing permanent teeth and hypohidrosis, his skin and nails were normal (Fig. 1 and Table 1). The @VARIANT$ (c.457C>T) mutation was found in exon 3 of EDA, it results in the substitution of Arg at residue 153 to Cys. Moreover, a heterozygous p.Gly213Ser (c.637G>A) mutation was detected in exon 3 of @GENE$, this leads to the substitution of Gly at residue 213 to Ser.

EDA;1896

WNT10A;22525

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

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

0no label

Deleterious variants in HS1BP3 (NM_022460.3: c.94C>A, @VARIANT$) and GNA14 (NM_004297.3: @VARIANT$, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP. Deleterious variants in DNAH17,TRPV4,CAPN11,VPS13C,UNC13B,@GENE$,@GENE$, and MRPL15 were found in two or more independent pedigrees.

SPTBN4;11879

MYOD1;7857

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

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

0no label

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

EDNRB;89

SOX10;5055

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

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

0no label

Three variants in three genes were rare, including the @GENE$ gene mutation [p.(Lys205del)], a novel heterozygous missense variant [@VARIANT$; p.(@VARIANT$)] in the SEMA7A gene (NM_001146029), as well as a splice site variation in the @GENE$ gene (NM_032242; MAF = 0.03 in GnomAD).

PROKR2;16368

PLXNA1;56426

c.1801G > A;tmVar:c|SUB|G|1801|A;HGVS:c.1801G>A;VariantGroup:2;CorrespondingGene:8482;RS#:750920992;CA#:7656750

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

0no label

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

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

25 The RYR3 (NM_001036: c.7812C > G, p.Asn2604Lys) and @GENE$ (NM_001159936: c.1034A > T, p.Asn345Ile) variants were classified as likely benign and benign, respectively, while the @GENE$ (NM_003302: c.822G > C, @VARIANT$) and the CAPN9 (NM_006615: c.55G > T, @VARIANT$) variants were classified as VUS.

EBNA1BP2;4969

TRIP6;37757

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

p.Ala19Ser;tmVar:p|SUB|A|19|S;HGVS:p.A19S;VariantGroup:17;CorrespondingGene:10753;RS#:147360179;CA#:1448452

0no label

The proband is heterozygous for the @GENE$/TACI @VARIANT$ mutation and meets the Ameratunga et al. diagnostic criteria for CVID and the American College of Rheumatology criteria for systemic lupus erythematosus (SLE). Her son has type 1 diabetes, arthritis, reduced IgG levels and IgA deficiency, but has not inherited the TNFRSF13B/TACI mutation. Her brother, homozygous for the TNFRSF13B/TACI mutation, is in good health despite profound hypogammaglobulinemia and mild cytopenias. We hypothesised that a second unidentified mutation contributed to the symptomatic phenotype of the proband and her son. Whole-exome sequencing of the family revealed a de novo nonsense mutation (@VARIANT$) in the @GENE$ (TCF3) gene encoding the E2A transcription factors, present only in the proband and her son.

TNFRSF13B;49320

Transcription Factor 3;2408

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

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

11

Four potential pathogenic variants, including SCN5A p.R1865H (NM_001160160, c.G5594A), @GENE$ p.A961T (NM_000426, c.G2881A), KCNH2 p.307_308del (NM_001204798, c.921_923del), and DMD p.E1028V (NM_004011, @VARIANT$) were involved in the occurrence of arrhythmia and cardiomyopathy (Table 2). In these known and candidate genes, @GENE$ gene encodes voltage-gated potassium channel activity of cardiomyocytes, which participated in the action potential repolarization. SCN5A gene encodes for voltage-gated sodium channel subunit as an integral membrane protein, responsible for the initial upstroke of the action potential (obtained from GenBank database). Mutations of KCNH2 and SCN5A genes are closely related to LQTS. The mutations of KCNH2 p.307_308del and SCN5A p.R1865H were found in the proband by WES and validated as positive by Sanger sequencing. Additionally, the heterozygous SCN5A @VARIANT$ was carried by I: 1 and II: 2, but not carried by I: 2 (Figure 1a).

LAMA2;37306

KCNH2;201

c.A3083T;tmVar:c|SUB|A|3083|T;HGVS:c.3083A>T;VariantGroup:5;CorrespondingGene:3757

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

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; g.146466A>G, c.4333A>G, @VARIANT$) and one in @GENE$ (@VARIANT$, c.637G>A, p.Gly213Ser) (Figure 2A and Figure S2A,B).

LRP6;1747

WNT10A;22525

p.Met1445Val;tmVar:p|SUB|M|1445|V;HGVS:p.M1445V;VariantGroup:6;CorrespondingGene:4040;RS#:761703397

g.14712G>A;tmVar:g|SUB|G|14712|A;HGVS:g.14712G>A;VariantGroup:7;CorrespondingGene:80326;RS#:147680216;CA#:211313

11

Two nucleotide variants in exon 8 (c.868 G > T; @VARIANT$) of the GCK gene and in exon 4 (@VARIANT$; p.Pro291Arg) of the HNF1A gene were identified. These variants were confirmed with standard Sanger sequencing. Molecular sequencing extended to the diabetic parents showed that the @GENE$ variant was present in the father and the @GENE$ variant was present in the mother (Figure 1B).

GCK;55440

HNF1A;459

p.Glu290*;tmVar:p|SUB|E|290|*;HGVS:p.E290*;VariantGroup:9;CorrespondingGene:2645

c.872 C > G;tmVar:c|SUB|C|872|G;HGVS:c.872C>G;VariantGroup:2;CorrespondingGene:6927;RS#:193922606;CA#:214336

11

We identified four genetic variants (KCNQ1-p.R583H, KCNH2-@VARIANT$, @GENE$-p.K897T, and @GENE$-@VARIANT$) in an LQTS family.

KCNH2;201

KCNE1;3753

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

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

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 @VARIANT$ of @GENE$, resulting in an asparagine into serine substitution in codon 166 (N166S) and for the @VARIANT$ of GJB2 (Fig. 1b, d).

GJB2;2975

GJB3;7338

A to G transition at nucleotide position 497;tmVar:c|SUB|A|497|G;HGVS:c.497A>G;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

Patient P0432 has a c.4030_4037delATGGCTGG (@VARIANT$) mutation in USH2A and a missense mutation in @GENE$ (@VARIANT$), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (c.46C>G; p.L16V) and @GENE$ (c.9921T>G).

CDH23;11142

USH2A;66151

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

0no label

The @VARIANT$ (c.936C>G) mutation in @GENE$ and heterozygous p.Arg171Cys (c.511C>T) mutation in WNT10A were detected. The coding sequence in exon 9 of EDA showed a C to G transition, which results in the substitution of Ile at residue 312 to Met; also, the coding sequence in exon 3 of WNT10A showed a @VARIANT$, which results in the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome revealed that the mutant alleles were from his mother, who carried digenic heterozygous EDA and @GENE$ mutations at the same locus as that of N2 (Fig. 2B).

EDA;1896

WNT10A;22525

p.Ile312Met;tmVar:p|SUB|I|312|M;HGVS:p.I312M;VariantGroup:7;CorrespondingGene:1896

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

We transfected wild-type or mutant plasmids into HEK293T cells and found that some FLNB variants (including p.M1803L, p.S2503G and p.T2166M; online supplementary figure 1) resulted in cytoplasmic focal accumulation, and some other FLNB variants (including p.R566L, p.A2282T, p.S2503G, @VARIANT$ and @VARIANT$; online supplementary figure 2) altered actin dynamics (online supplementary figures 1 and 2). @GENE$ and @GENE$ variants in individuals with AIS. (A) Profiles of rare damaging variants in FLNB.

FLNB;37480

TTC26;11786

p.R199Q;tmVar:p|SUB|R|199|Q;HGVS:p.R199Q;VariantGroup:0;CorrespondingGene:79989;RS#:1175244100

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

0no label

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

WNT10A;22525

EDA;1896

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

We identified a novel compound heterozygous variant in BBS1 c.1285dup (p.(Arg429Profs*72); a likely pathogenic novel variant affecting the conserved residue 354 in the functional domain of @GENE$ (@VARIANT$; p.(Asn354Lys)); a pathogenic new homozygous nucleotide change in BBS7 that leads to a @VARIANT$, c.763A > T, and a likely pathogenic homozygous substitution c.1235G > T in @GENE$, leading to the change p.(Cys412Phe).

BBS2;12122

BBS6;10318

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

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

0no label

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 @GENE$ @VARIANT$ 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. Two SALS patients carried multiple ALS-associated variants that are rare in population databases (@GENE$ @VARIANT$ with VAPB p.M170I and TAF15 p.R408C with SETX p.I2547T and SETX p.T14I).

VAPB;36163

ANG;74385

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

p.K41I;tmVar:p|SUB|K|41|I;HGVS:p.K41I;VariantGroup:28;CorrespondingGene:283;RS#:1219381953

0no label

Interestingly, we identified 5 patients (4.8%) with variants in optineurin (@GENE$) and TANK-binding kinase 1 (@GENE$) that are predicted to be highly pathogenic, including two double mutants. Case A was a compound heterozygote for mutations in OPTN, carrying the @VARIANT$ nonsense and p.A481V missense mutation in trans, while case B carried a deletion of OPTN exons 13-15 (p.Gly538Glufs*27) and a loss-of-function mutation (@VARIANT$) in TBK1.

OPTN;11085

TBK1;22742

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

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

0no label

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

SOX10;5055

MITF;4892

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

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

0no label

We observed that in 5 PCG cases heterozygous @GENE$ mutations (@VARIANT$, p.E229 K, and p.R368H) co-occurred with heterozygous @GENE$ mutations (p.E103D, @VARIANT$, p.Q214P, and p.G743A) indicating a potential digenic inheritance (Fig. 1a).

CYP1B1;68035

TEK;397

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

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

11

Given the reported normal function of pendrin L117F and pendrin S166N as an anion exchanger, compromised regulatory machinery of @GENE$ 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$, and pendrin F355L mutations on EphA2 interaction and internalization was examined. While the amount of co-precipitated pendrin mutants with @GENE$ 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 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 p.T511M and SLC26A4 @VARIANT$ mutations.

pendrin;20132

EphA2;20929

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

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

0no label

Amino acid conservation analysis showed that seven of the 10 variants (CELSR1 p.G1122S, @GENE$ @VARIANT$, DVL3 p.R148Q, PTK7 p.P642R, SCRIB p.G1108E, SCRIB p.G644V and @GENE$ @VARIANT$) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

CELSR1;7665

SCRIB;44228

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

p.K618R;tmVar:p|SUB|K|618|R;HGVS:p.K618R;VariantGroup:2;CorrespondingGene:5754;RS#:139041676

0no label

Given the reported normal function of pendrin @VARIANT$ and pendrin S166N as an anion exchanger, compromised regulatory machinery of pendrin function may cause the observed symptoms. To examine whether @GENE$ is involved in dysfunction of pendrin caused by these amino acid substitutions, the effect of pendrin L117F, pendrin S166N, 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 @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 p.T511M and SLC26A4 @VARIANT$ mutations.

EphA2;20929

ephrin-B2;3019

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

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

0no label

We report digenic variants in SCRIB and PTK7 associated with NTDs in addition to SCRIB and @GENE$ heterozygous variants in additional NTD cases. The combinatorial variation of PTK7 @VARIANT$ (p.P642R) and SCRIB c.3323G > A (p.G1108E) only occurred in one spina bifida case, and was not found in the 1000G database or parental samples of NTD cases. Location analysis of missense changes showed that @VARIANT$ was located very close to the fourth PDZ domain (1109-1192) of @GENE$. The PDZ domains of human SCRIB are required for correct localization and physical interaction with other proteins, such as the core PCP protein VANGL2, which is required for transducing PCP signals.

CELSR1;7665

SCRIB;44228

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

Exome analysis for the proband identified three sequence variants in FTA candidate genes, two in @GENE$ (g.27546T>A, @VARIANT$, p.Ser127Thr; g.124339A>G, c.3224A>G, p.Asn1075Ser) and one in @GENE$ (@VARIANT$, c.499G>C, p.Glu167Gln) (Figure 4A).

LRP6;1747

WNT10A;22525

c.379T>A;tmVar:c|SUB|T|379|A;HGVS:c.379T>A;VariantGroup:1;CorrespondingGene:4040;RS#:17848270;CA#:6455897

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

11

We observed that in 5 PCG cases heterozygous CYP1B1 mutations (@VARIANT$, p.E229 K, and p.R368H) co-occurred with heterozygous TEK mutations (@VARIANT$, 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 @GENE$ and TEK mutations. The TEK Q214P and G743A alleles were absent in 1024 controls, whereas very low frequencies of heterozygous @GENE$ 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

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

0no label

Results Cosegregating deleterious variants (GRCH37/hg19) in @GENE$ (NM_001127222.1: @VARIANT$, @VARIANT$), REEP4 (NM_025232.3: c.109C>T, p.Arg37Trp), TOR2A (NM_130459.3: c.568C>T, 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

p.Pro2421Val;tmVar:p|SUB|P|2421|V;HGVS:p.P2421V;VariantGroup:3;CorrespondingGene:80346

0no label

It was shown that digenic variants in @GENE$ and @GENE$ contribute to PCG and that variants in both FOXC1 and PITX2 are responsible for some cases of ARS. This prompted us to explore the frequency of CHD in patients with ARS carrying a Foxc1 mutation and whether or not there is a need to carry on WES to investigate the role of other variants in conjunction with FOXC1 that would explain these cardiac defects. Whole Exome Sequencing A tool to draw genotype-phenotype correlation out of the 67 FOXC1 variants reported so far to be linked to the ARS, only nine have been shown to be linked to cardiac defects in addition to the ocular defects. A scrutinized review of the literature of these nine variants, namely @VARIANT$, p.P79T, p.S82T, p. A85P, @VARIANT$, p.F112S, p.R127L, p.G149D, and p.R170W, did show that the cardiac phenotype with which they are associated is not as clear as it is presumed.

CYP1B1;68035

MYOC;220

p.Q70Hfs*8;tmVar:p|FS|Q|70|H|8;HGVS:p.Q70HfsX8;VariantGroup:8;CorrespondingGene:6012

p.L86F;tmVar:p|SUB|L|86|F;HGVS:p.L86F;VariantGroup:6;CorrespondingGene:2296;RS#:886039568;CA#:10588416

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: @GENE$/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; @VARIANT$ of KAL1) and NELF/@GENE$ (c. 1160-13C>T of NELF and @VARIANT$; p.Trp275X of TACR3).

NELF;10648

TACR3;824

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

c.824G>A;tmVar:c|SUB|G|824|A;HGVS:c.824G>A;VariantGroup:1;CorrespondingGene:26012;RS#:144292455;CA#:144871

0no label

These seven amino acids are located in the peroxidase- (PO-) like domain and are conserved among @GENE$ orthologs (Figure 2 and Figure S1). The latter variant likely resulted in aberrant splicing of the transcript. Two novel variants were identified in TG, including one frameshift mutation (c.2060_2060delG, p.C687LfsX34) and one missense mutation (c.1514G>A, @VARIANT$). A novel missense mutation was found in @GENE$ (c.398G>A, @VARIANT$).

DUOX2;9689

DUOXA2;57037

p.G505D;tmVar:p|SUB|G|505|D;HGVS:p.G505D;VariantGroup:10;CorrespondingGene:7173;RS#:867829370

p.R133H;tmVar:p|SUB|R|133|H;HGVS:p.R133H;VariantGroup:16;CorrespondingGene:7038;RS#:745463507;CA#:4885341

0no label

The nucleotide sequence showed a G to C transition at nucleotide 769 (@VARIANT$) of the coding sequence in exon 7 of @GENE$, 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$) of the coding sequence in exon 3 of @GENE$, which results in the substitution of Arg at residue 171 to Cys.

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

The coding sequence in exon 9 of @GENE$ showed a C to G transition, which results in the substitution of @VARIANT$; also, the coding sequence in exon 3 of @GENE$ showed a @VARIANT$, which results in the substitution of Arg at residue 171 to Cys.

EDA;1896

WNT10A;22525

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

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

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 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. @GENE$ variants have been mostly detected in familial ALS cases that are localized within the C-terminus of the FUS protein.

SPG11;41614

FUS;2521

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

Amino acid conservation analysis showed that seven of the 10 variants (CELSR1 p.G1122S, @GENE$ @VARIANT$, DVL3 p.R148Q, PTK7 p.P642R, @GENE$ @VARIANT$, SCRIB p.G644V and SCRIB p.K618R) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

CELSR1;7665

SCRIB;44228

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

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

0no label

To investigate the role of GJB3 variations along with GJB2 mutations for a possible combinatory allelic disease inheritance, we have screened patients with heterozygous @GENE$ mutations for variants in @GENE$ by sequencing. Analysis of the entire coding region of the Cx31 gene revealed the presence of two different missense mutations (N166S and @VARIANT$) occurring in compound heterozygosity along with the 235delC and 299delAT of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and @VARIANT$/A194T).

GJB2;2975

Cx31;7338

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

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

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$. Additionally, the nucleotide sequence showed a monoallelic @VARIANT$ (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 @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

Gly at residue 257 to Arg;tmVar:p|SUB|G|257|R;HGVS:p.G257R;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

In patient AVM144, the compound heterozygous variants c.116-1G>A and @VARIANT$ (p.Ser334Thr) were identified in PTPN13 (table 2). Potential oligogenic inheritance Variants in more than one gene (at least one likely pathogenic variant) with differing inheritance origin were identified in three patients (figure 1). In patient AVM558, a pathogenic heterozygous variant c.920dupA (p.Asn307LysfsTer27) inherited from the mother was identified in @GENE$. Another de novo novel heterozygous missense variant, c.1694G>A (@VARIANT$), was identified in @GENE$ (online supplementary table S2), which encodes the kinase responsible for phosphorylation of residue T312 in SMAD1 to block its activity in BMP/TGF-beta signalling.

ENG;92

MAP4K4;7442

c.1000T>A;tmVar:c|SUB|T|1000|A;HGVS:c.1000T>A;VariantGroup:0;CorrespondingGene:5783;RS#:755467869;CA#:2995566

p.Arg565Gln;tmVar:p|SUB|R|565|Q;HGVS:p.R565Q;VariantGroup:5;CorrespondingGene:9448;RS#:1212415588

0no label

Six variants in @GENE$ occurred de-novo, three of which were not previously described: c.3236del p.(Asp1079Alafs*25), @VARIANT$ p.(Glu2954*), and c.9201+1G>A. One de-novo and novel variant was also detected in @GENE$: c.992G>A p.(@VARIANT$).

PKD1;250

PKD2;20104

c.8860G>T;tmVar:c|SUB|G|8860|T;HGVS:c.8860G>T;VariantGroup:46;CorrespondingGene:5310

Cys331Tyr;tmVar:p|SUB|C|331|Y;HGVS:p.C331Y;VariantGroup:1;CorrespondingGene:23193;RS#:144118755

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 PKHD1: c.1675C > T (p.R559W) and PKHD1: c.7942G > A (@VARIANT$), which were inherited from the mother and the father, respectively (Figure 3). Whole-exome sequencing revealed that the foetal PKD proband in Family 23 had compound heterozygous variants in @GENE$, which was subsequently verified through Sanger sequencing.

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

Direct sequence analysis showing the @VARIANT$ mutation (l) and wild type (WT) allele (m) of GJB2. Direct sequence analysis showing the 497A>G (N166S) mutation (d) and WT allele (e) of GJB3. Direct sequence analysis showing the @VARIANT$ (A194T) mutation (i and n) and WT allele (j and o) of GJB3. Expression of Cx31 and @GENE$ in the mouse cochlea examined by coimmunostaining Cochlear cryosections were cut at a thickness of 8 mum and labeled with an antibody against Cx26 (a) and @GENE$ (b).

Cx26;2975

Cx31;7338

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

580G>A;tmVar:c|SUB|G|580|A;HGVS:c.580G>A;VariantGroup:4;CorrespondingGene:2707;RS#:117385606;CA#:118313

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$. Additionally, the nucleotide sequence showed a monoallelic @VARIANT$ (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 @GENE$ 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 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

DISCUSSION In this study, we describe identification and characterization of abnormalities in patients with homozygous mutations in two genes, a novel mutation in @GENE$, @VARIANT$ and a previously identified mutation in @GENE$, @VARIANT$. The affected patients presented with moderate global developmental delay, tall stature, obesity, macrocephaly, mild dysmorphic features, hypertelorism, maloccluded teeth, intellectual disability, and flat feet.

SEC23A;4642

MAN1B1;5230

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

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

11

c.223 - 4C > A might affect the normal splicing of exons in the PROK2 gene, and the novel variant @VARIANT$ (p. Arg102Ser) was predicted to be harmful by multiple software programs. A few missense variants were detected in patients with a PROK2 gene, and most of the missense variants recorded in the ClinVar database were pathogenic. Three kinds of missense variants in the @GENE$ gene were found in eight patients. c.337 T > C (p. Tyr113His) significantly decreased the receptor expression level and reduced intracellular calcium mobilization, resulting in protein instability and poor biological function. c.491G > A (p. Arg164Gln) destroyed the interaction between the IL2 domain and G-protein, inhibited Gq-protein signal activity, and weakened G protein-coupled receptors. The hot spot variant @VARIANT$ (p. Trp178Ser) was found in six patients and located in the transmembrane domain of the protein, which could significantly reduce the release of ionized calcium and the signal activity. The FGFR1 gene is expressed in many tissues and plays an important role in the development of embryonic olfactory nerve and GnRH neurons mainly through the FGF/@GENE$ signalling pathway.

PROKR2;16368

FGFR1;69065

c.306G > C;tmVar:c|SUB|G|306|C;HGVS:c.306G>C;VariantGroup:27;CorrespondingGene:60675

c.533G > C;tmVar:c|SUB|G|533|C;HGVS:c.533G>C;VariantGroup:12;CorrespondingGene:128674;RS#:201835496;CA#:270917

0no label

Myopathy With @GENE$ and @GENE$ Variants: Clinical and Pathological Features Objective The aim of this study is to identify the molecular defect of three unrelated individuals with late-onset predominant distal myopathy; to describe the spectrum of phenotype resulting from the contributing role of two variants in genes located on two different chromosomes; and to highlight the underappreciated complex forms of genetic myopathies. Patients and methods Clinical and laboratory data of three unrelated probands with predominantly distal weakness manifesting in the sixth-seventh decade of life, and available affected and unaffected family members were reviewed. Next-generation sequencing panel, whole exome sequencing, and targeted analyses of family members were performed to elucidate the genetic etiology of the myopathy. Results Genetic analyses detected two contributing variants located on different chromosomes in three unrelated probands: a heterozygous pathogenic mutation in SQSTM1 (c.1175C>T, @VARIANT$) and a heterozygous variant in TIA1 (c.1070A>G, @VARIANT$).

SQSTM1;31202

TIA1;20692

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

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

11

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 c.730C>G), 2F07 (CELSR1 c.8807C>T and @GENE$ 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 c.1531C>T and CELSR2 c.3800A>G), 335F07 (@GENE$ c.544G>A and 2 FAT4 missense variants @VARIANT$; @VARIANT$), and 465F99 (rare FZD1 missense variant c.211C>T and a novel FAT4 missense variant c.10147G>A).

DVL3;20928

FZD6;2617

c.5792A>G;tmVar:c|SUB|A|5792|G;HGVS:c.5792A>G;VariantGroup:2;CorrespondingGene:79633;RS#:373263457;CA#:4677776

c.10384A>G;tmVar:c|SUB|A|10384|G;HGVS:c.10384A>G;VariantGroup:2;CorrespondingGene:4824;RS#:373263457;CA#:4677776

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

EDA;1896

WNT10A;22525

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

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

0no label

Similarly, the CCDC88C-mutated case P05 in our study carried additional variants in @GENE$ (DCC)p. Gln91Arg, and FGFR1 @VARIANT$, implying that the deleterious variants in CCDC88C act together with other variants to cause IHH through a digenic/oligogenic model. One unreported and probably deleterious missense variant p. Val969Ile of another PSIS gene, CDON, was also found in case P17 who carried a missense variant in CHD7, a causative gene of IHH. CDON seems to act similarly as @GENE$ through a digenic/oligogenic model to contribute to IHH. Case P06 had a missense variant in GADL1 (@VARIANT$), predicted as probably damaging.

DCC netrin 1 receptor;21081

CCDC88C;18903

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

p. Ser221Cys;tmVar:p|SUB|S|221|C;HGVS:p.S221C;VariantGroup:5;CorrespondingGene:339896;RS#:775162663;CA#:2294666

0no label

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

EDNRB;89

PAX3;22494

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

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. @GENE$ encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, S275N) were detected. The @VARIANT$ variant affects the b isoform of the MATR3 protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms. 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

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

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

0no label