Datasets:

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The T338I and @VARIANT$ variants affect the conserved central coiled-coil rod domain of the protein mediating dimerization; therefore, we suggest their potential deleterious effect on the protein. In the individual carrying the P505L NEFH variant, an additional novel alteration (C335R) was detected in the @GENE$ gene. Loss-of-function GRN variants are primarily considered to cause frontotemporal lobar degeneration, but there is evidence that missense GRN variants are also linked to the pathogenesis of ALS. The novel GRN variant reported in this study results in a cysteine-to-arginine change in the cysteine-rich granulin A domain. Four cases were identified to carry SQSTM1 variants: the P392L in two cases and the @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

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

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

0no label

c, d) Sequence chromatograms indicating the wild-type, homozygous affected and heterozygous carrier forms of c) the C to T transition at position c.229 changing the arginine residue to cysteine at position 77 of the @GENE$ protein (@VARIANT$; p.R77C) and d) the @VARIANT$ (p.I80Gfs*13) in S100A13. Mutation name is based on the full-length S100A3 (NM_002960) and @GENE$ (NM_001024210) transcripts.

S100A3;2223

S100A13;7523

c.229C>T;tmVar:c|SUB|C|229|T;HGVS:c.229C>T;VariantGroup:3;CorrespondingGene:6274;RS#:138355706;CA#:1116284

c.238-241delATTG;tmVar:c|DEL|238_241|ATTG;HGVS:c.238_241delATTG;VariantGroup:13;CorrespondingGene:6284

0no label

We identified four genetic variants (@GENE$-@VARIANT$, KCNH2-p.C108Y, @GENE$-p.K897T, and KCNE1-@VARIANT$) in an LQTS family.

KCNQ1;85014

KCNH2;201

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

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

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/@GENE$ (@VARIANT$; p.Ala253Thr of NELF and c.488_490delGTT; p.Cys163del of KAL1) and @GENE$/TACR3 (c. 1160-13C>T of NELF and c.824G>A; @VARIANT$ of TACR3).

KAL1;55445

NELF;10648

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

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

0no label

In a second example, we identified a monoallelic change in @GENE$ (c.G680A, p.Arg227Gln, @VARIANT$:G>A), in conjunction with the @VARIANT$ of @GENE$. Monoallelic inheritance of SRD5A2, although uncommon, has been reported in a severely under-virilized individual with hypospadias and bilateral inguinal testes (Chavez, Ramos, Gomez, & Vilchis, 2014).

SRD5A2;37292

SF1;138518

rs9332964;tmVar:rs9332964;VariantGroup:0;CorrespondingGene:6716;RS#:9332964

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

11

To investigate the role of @GENE$ 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 @VARIANT$ 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 GJB3, resulting in an asparagine into serine substitution in codon 166 (N166S) and for the 235delC of GJB2 (Fig. 1b, d).

GJB3;7338

GJB2;2975

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

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

0no label

In the subject III.1, the variant, carried in the heterozygous status, is the c.868 G > T; @VARIANT$, in the glucokinase (@GENE$) gene; the III.2 subject carried the @VARIANT$; p.Pro291Arg, in the @GENE$ gene.

CGK;55964

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

0no label

The nucleotide sequence showed a G to C transition at nucleotide 769 (@VARIANT$) of the coding sequence in exon 7 of EDA, which results in the substitution of Gly at residue 257 to Arg. Additionally, the nucleotide sequence showed a monoallelic @VARIANT$ (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 @GENE$ mutation (c.769G>C) and a heterozygous WNT10A c.511C>T mutation, and showed absence of only the left upper lateral incisor without other clinical abnormalities. No mutations in these genes were found in the father. Sequence analyses of EDA and @GENE$ genes.

EDA;1896

WNT10A;22525

c.769G>C;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

Both homozygous and compound heterozygous variants in the @GENE$ gene have been described as causative for juvenile ALS. The G1177X nonsense variant was first detected in compound heterozygous form in a family with two affected siblings suffering from infantile ascending spastic paralysis with bulbar involvement. The ages of onset of the patients with the ALS2 variants reported in this study were later than juvenile ALS onset, which generally manifests before 25 years of age. Previous studies suggested that heterozygous variants in the ALS2 may be causative for adult-onset sALS. @GENE$ encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, 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 (@VARIANT$ and G4290R) in the DYNC1H1 gene.

ALS2;23264

MATR3;7830

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

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

0no label

Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A @VARIANT$ (p.M400I) and @GENE$ c.1000C>T (@VARIANT$), associated with congenital birth defects in two patients from a consanguineous family. Patients presented with carbohydrate-deficient transferrin, tall stature, obesity, macrocephaly, and maloccluded teeth. The parents were healthy heterozygous carriers for both mutations and an unaffected sibling with tall stature carried the heterozygous mutation in @GENE$ only.

MAN1B1;5230

SEC23A;4642

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

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

0no label

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

KAL1;55445

NELF;10648

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

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, @VARIANT$, c.494C>T, c.3719G>A and c.5749G>T in @GENE$, c.238_239dupC in USH1C, and @VARIANT$ and c.10712C>T 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.1556G>A;tmVar:c|SUB|G|1556|A;HGVS:c.1556G>A;VariantGroup:9;CorrespondingGene:4647;RS#:111033206;CA#:278629

c.2299delG;tmVar:c|DEL|2299|G;HGVS:c.2299delG;VariantGroup:190;CorrespondingGene:7399;RS#:80338903

0no label

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

KAL1;55445

NELF;10648

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

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

0no label

In patient AVM226, we identified the compound heterozygous variants @VARIANT$ (p.Val1259Ile) and c.2966A>T (@VARIANT$) in @GENE$ (table 2). @GENE$ and DSCAM have similar neurodevelopmental functions and are essential for self-avoidance in the developing mouse retina.

DSCAM;74393

DSCAML1;79549

c.3775G>A;tmVar:c|SUB|G|3775|A;HGVS:c.3775G>A;VariantGroup:5;CorrespondingGene:1826;RS#:1212415588

p.Gln989Leu;tmVar:p|SUB|Q|989|L;HGVS:p.Q989L;VariantGroup:5;CorrespondingGene:83394;RS#:1212415588

0no label

On the other hand, two missense mutations of the EPHA2 gene were identified in two families, SLC26A4: c.1300G>A (p.434A>T), EPHA2: c.1063G>A (@VARIANT$) and @GENE$: @VARIANT$ (p.410T>M), @GENE$: c.1532C>T (p.T511M) (Fig. 6a, b).

SLC26A4;20132

EPHA2;20929

p.G355R;tmVar:p|SUB|G|355|R;HGVS:p.G355R;VariantGroup:4;CorrespondingGene:1969;RS#:370923409;CA#:625329

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

0no label

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

NELF;10648

KAL1;55445

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

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

0no label

Two different GJB3 mutations (@VARIANT$ and A194T) occurring in compound heterozygosity with the 235delC and @VARIANT$ of @GENE$ were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in @GENE$ was detected in DNA from 200 unrelated Chinese controls.

GJB2;2975

Cx31;7338

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

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

0no label

Interestingly, four of these TEK mutations (p.E103D, @VARIANT$, p.Q214P, and p.G743A) co-occurred with three heterozygous mutations in another major PCG gene @GENE$ (p.A115P, p.E229K, and @VARIANT$) in five families. The parents of these probands harbored either of the heterozygous @GENE$ or CYP1B1 alleles and were asymptomatic, indicating a potential digenic mode of inheritance.

CYP1B1;68035

TEK;397

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

On the basis of the data collected in this study, we may speculate that the presence of @GENE$-@VARIANT$, together with three @GENE$-@VARIANT$ alleles, could lead to an increased risk of developing cardiac arrhythmias due to the prolongation of the QT interval.

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

11

Moreover, patients carrying a LAMA4 @VARIANT$ 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 @GENE$ (L1038P) and @GENE$ (@VARIANT$), both encoding sarcomeric proteins that are likely to affect its structure when mutated.

MYH7;68044

MYBPC3;215

Pro943Leu;tmVar:p|SUB|P|943|L;HGVS:p.P943L;VariantGroup:5;CorrespondingGene:3910;RS#:387907365;CA#:143749

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

0no label

Moreover, this MITF variant was not detected in the 666 control chromosomes from normal hearing Korean subjects, supporting the pathogenic potential of p.R341C in @GENE$ in SH107-225. However, symptoms and signs suggesting Waardenburg syndrome type2 (WS2) including retinal abnormalities and pigmentation abnormalities could not be determined due of the patients' young ages. Digenic inheritances of GJB2/MITF and GJB2/@GENE$ (group II). (A) In addition to c.235delC 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. (C) The sequence of the p.R341C 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.

MITF;4892

GJB3;7338

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

In patient AVM226, we identified the compound heterozygous variants c.3775G>A (p.Val1259Ile) and c.2966A>T (p.Gln989Leu) in @GENE$ (table 2). DSCAML1 and DSCAM have similar neurodevelopmental functions and are essential for self-avoidance in the developing mouse retina. In patient AVM144, the compound heterozygous variants @VARIANT$ and c.1000T>A (p.Ser334Thr) were identified in @GENE$ (table 2). Potential oligogenic inheritance Variants in more than one gene (at least one likely pathogenic variant) with differing inheritance origin were identified in three patients (figure 1). In patient AVM558, a pathogenic heterozygous variant c.920dupA (p.Asn307LysfsTer27) inherited from the mother was identified in ENG. Another de novo novel heterozygous missense variant, c.1694G>A (@VARIANT$), was identified in MAP4K4 (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.

DSCAM;74393

PTPN13;7909

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

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

0no label

Results Family with inherited neutropaenia, monocytosis and hearing impairment associated with mutations in GFI1 and @GENE$. Pedigree, phenotypes and mutation status are indicated as per the key provided (a). Causative heterozygous mutations in @GENE$ (p.N382S/@VARIANT$) and MYO6 (@VARIANT$/c.3526A > C) were identified by whole exome sequencing performed on III-1 and IV-1.

MYO6;56417

GFI1;3854

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

p.I1176L;tmVar:p|SUB|I|1176|L;HGVS:p.I1176L;VariantGroup:2;CorrespondingGene:4646;RS#:755922465;CA#:141060203

0no label

The @VARIANT$ variant in GJB2 occurring in complex heterozygosity with a pathogenic @GENE$ variant, @VARIANT$ from SH175-389, suggests a possible digenic etiology of SNHL involving two different gap junction proteins, @GENE$ and Cx31.

GJB3;7338

Cx26;2975

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

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

0no label

Four genes (including @GENE$, @GENE$, SCAP, TCF4) were found to be related to the PMI related. It turned out to be that only SCAP-c.3035C>T (@VARIANT$) and AGXT2-c.1103C>T (@VARIANT$) were predicted to be causive by both strategies.

AGXT2;12887

ZFHX3;21366

p.Ala1012Val;tmVar:p|SUB|A|1012|V;HGVS:p.A1012V;VariantGroup:2;CorrespondingGene:22937

p.Ala338Val;tmVar:p|SUB|A|338|V;HGVS:p.A338V;VariantGroup:5;CorrespondingGene:64902

0no label

In the subject III.1, the variant, carried in the heterozygous status, is the c.868 G > T; @VARIANT$, in the @GENE$ (CGK) gene; the III.2 subject carried the c.872 C > G; @VARIANT$, in the @GENE$ gene.

glucokinase;55440

HNF1A;459

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

p.Pro291Arg;tmVar:p|SUB|P|291|R;HGVS:p.P291R;VariantGroup:2;CorrespondingGene:6927;RS#:193922606;CA#:214336

0no label

In patient AVM028, one novel heterozygous VUS (c.2207A>G [@VARIANT$]) in @GENE$ inherited from the father and one likely pathogenic de novo novel heterozygous variant (c.311T>C [@VARIANT$]) in TIMP3 were identified (online supplementary table S2). While TIMP3 blocks VEGF/@GENE$ signalling, RASA1 modulates differentiation and proliferation of blood vessel endothelial cells downstream of VEGF (figure 3).

RASA1;2168

VEGFR2;55639

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

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

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; @VARIANT$ of @GENE$) and @GENE$/TACR3 (c. 1160-13C>T of NELF and c.824G>A; @VARIANT$ of TACR3).

KAL1;55445

NELF;10648

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

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

0no label

To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and @GENE$: @VARIANT$, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and @VARIANT$; PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare @GENE$ and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.

PITX2;55454

FOXC2;21091

c.535C>A;tmVar:c|SUB|C|535|A;HGVS:c.535C>A;VariantGroup:3;CorrespondingGene:1545;RS#:771076928

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

0no label

To sum up, SH166-367, SH170-377, and SB175-334 which would have been considered DFNB1 without TES were found to be DFNB7/11, DFNB3, and @GENE$, respectively. Finally, a subject with the heterozygous @VARIANT$ mutation in @GENE$ (SH60-136) carried a @VARIANT$ variant in Wolfram syndrome 1 (WFS1) (NM_001145853) according to TES.

DFNB16;15401

GJB2;2975

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

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

0no label

CSS170323 carries a heterozygous missense variant @VARIANT$(p.Met210Ile) in MYOD1 and a heterozygous missense variant c.190G>A(@VARIANT$) in MEOX1 (Table 2). CSS170323 presented with L2 hemivertebra and fused ribs (the right 11th rib and 12th rib). During mesoderm development, the expression of MEOX1 is increased by @GENE$ (Gianakopoulos et al., 2011), suggesting that these two variant potentially result in the cumulative perturbation of @GENE$-mediated pathway.

MYOD1;7857

TBX6;3389

c.630G>C;tmVar:c|SUB|G|630|C;HGVS:c.630G>C;VariantGroup:9;CorrespondingGene:4654;RS#:749634841;CA#:5906491

p.Ala64Thr;tmVar:p|SUB|A|64|T;HGVS:p.A64T;VariantGroup:5;CorrespondingGene:4222;RS#:373680176;CA#:8592682

0no label

Patient P0432 has a c.4030_4037delATGGCTGG (p.M1344fsX42) mutation in @GENE$ and a missense mutation in @GENE$ (@VARIANT$), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (@VARIANT$; p.L16V) and USH2A (c.9921T>G).

USH2A;66151

CDH23;11142

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

c.46C>G;tmVar:c|SUB|C|46|G;HGVS:c.46C>G;VariantGroup:18;CorrespondingGene:124590;RS#:876657419;CA#:10576353

0no label

The ISG20L2 and @GENE$ variants were excluded based on their frequencies in normal population cohorts. Sanger sequencing of Family 1 showed that both rs138355706 in @GENE$ (@VARIANT$, missense causing a p.R77C mutation) and a 4 bp deletion in S100A13 (@VARIANT$ causing a frameshift p.I80Gfs*13) segregated completely with ILD in Family 1 based upon recessive inheritance (figure 2c and d), were in total linkage disequilibrium, and were present in a cis conformation.

SETDB1;32157

S100A3;2223

c.229C>T;tmVar:c|SUB|C|229|T;HGVS:c.229C>T;VariantGroup:3;CorrespondingGene:6274;RS#:138355706;CA#:1116284

c.238-241delATTG;tmVar:c|DEL|238_241|ATTG;HGVS:c.238_241delATTG;VariantGroup:13;CorrespondingGene:6284

0no label

Recently, Gifford et al., identified three missense variants in @GENE$ (@VARIANT$), MYH7 (Leu387Phe), and @GENE$ (@VARIANT$) in three offspring with childhood-onset cardiomyopathy (Gifford et al., 2019).

MKL2;40917

NKX2-5;1482;4824

Gln670His;tmVar:p|SUB|Q|670|H;HGVS:p.Q670H;VariantGroup:2;CorrespondingGene:57496

Ala119Ser;tmVar:p|SUB|A|119|S;HGVS:p.A119S;VariantGroup:0;CorrespondingGene:1482;RS#:137852684;CA#:120058

11

In AS patient IID27, the two mutations in COL4A5 and @GENE$ were inherited independently, likely indicating an in trans configuration. There is a splicing site mutation @VARIANT$ in @GENE$, inherited from her mother and a missense mutation @VARIANT$ (p. (Thr1474Met)) inherited from her father (Figure 1a).

COL4A4;20071

COL4A5;133559

c.1339 + 3A>T;tmVar:c|SUB|A|1339+3|T;HGVS:c.1339+3A>T;VariantGroup:23;CorrespondingGene:1287

c.4421C > T;tmVar:c|SUB|C|4421|T;HGVS:c.4421C>T;VariantGroup:14;CorrespondingGene:1286;RS#:201615111;CA#:2144174

0no label

The R171 and @VARIANT$ residues are in yellow. The 3D structure of EDA is shown in Figure 4. The G257 residue is located at the interface of two trimers. When G257R mutation happened, the side chain volume significantly enlarged, making it possible to form interaction with the R289 in adjacent trimer and abolish the stabilization of EDA. @VARIANT$ is located at the outer surface of the three monomers. An I312M mutation could affect the interactions of EDA with its receptors. Structure analysis of mutant residues in the three-dimensional EDA trimer. The @GENE$ trimer is shown as a ribbon with relevant side chains rendered in spheres. The G257 and I312 residues are in yellow and blue, respectively. The side chain of the R289 residue is represented by a colored stick. (A) The planform of the EDA trimer. (B) The side view of the EDA trimer. Discussion This is the first study to show that simultaneous WNT10A and EDA mutations could lead to tooth agenesis in the Chinese population. We found that six participants harbored digenic mutations in both @GENE$ and EDA: two of them had isolated oligodontia and the others had syndromic tooth agenesis.

EDA;1896

WNT10A;22525

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

I312;tmVar:p|Allele|I|312;VariantGroup:7;CorrespondingGene:1896

0no label

CVID, common variable immunodeficiency disorder; SLE, systemic lupus erythematosus; sIgAD, selective IgA deficiency; T1D, Type 1 Diabetes, sHGUS, symptomatic hypogammglobulinaemia of uncertain significance; WT, wild-type. (b) Electropherograms showing the T168fsX191 mutation of TCF3 and @VARIANT$ (c.310T>C) mutation of TACI gene in the proband II.2. The proband's son (III.1) has inherited the TCF3 @VARIANT$ mutation, but not the TNFRSF13B/@GENE$ C104R mutation. The proband's clinically unaffected daughter (III.2) has not inherited either mutation. The @GENE$ T168fsX191 mutation was absent in the proband's parents, indicating a de novo origin.

TACI;49320

TCF3;2408

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

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

0no label

The detected @VARIANT$ variant affects the nuclear localization signal 2 (amino acids 568-574) of the @GENE$ protein. A previously characterized pathogenic nonsense variant (G1177X) and a rare missense alteration (@VARIANT$) were detected in the ALS2 gene, both in heterozygous form. The alsin protein encoded by the ALS2 gene is involved in endosome/membrane trafficking and fusion, cytoskeletal organization, and neuronal development/maintenance. Both homozygous and compound heterozygous variants in the ALS2 gene have been described as causative for juvenile ALS. The G1177X nonsense variant was first detected in compound heterozygous form in a family with two affected siblings suffering from infantile ascending spastic paralysis with bulbar involvement. The ages of onset of the patients with the ALS2 variants reported in this study were later than juvenile ALS onset, which generally manifests before 25 years of age. Previous studies suggested that heterozygous variants in the @GENE$ may be causative for adult-onset sALS.

CCNF;1335

ALS2;23264

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

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

0no label

The proband in family PCG-139 also carried a rare PITX2 variant (@VARIANT$) and presented glaucoma diagnosed at the age of seven days. Both probands required more surgical operations to control IOP than the rest of patients. Below symbols are indicated genotypes for CYP1B1 and PITX2, age at diagnosis and number or surgical operations per eye, respectively. M1, CYP1B1: p.(A179fs*18). M2, CYP1B1: p.(E387K). M3, CYP1B1: p.(E173*). M4, PITX2: @VARIANT$. M5, PITX2: p.(A188T). Arrows show the index cases. +: wild-type allele. The asterisk indicates a de novo PITX2 variant. Evolutionary conservation of @GENE$ and PITX2 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. This analysis revealed that most FOXC2 and @GENE$ wild-type amino acids or nucleotides were highly conserved (Fig 4).

FOXC2;21091

PITX2;55454

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

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

0no label

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

ALS2;23264

MATR3;7830

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

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

0no label

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

MITF;4892

TYRO3;4585

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

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

0no label

In this study, we performed whole-genome sequencing in 104 pathologically confirmed FTLD-TDP patients from the Mayo Clinic brain bank negative for @GENE$ and @GENE$ mutations and report on the contribution of rare single nucleotide and copy-number variants in 21 known neurodegenerative disease genes. Interestingly, we identified 5 patients (4.8%) with variants in optineurin (OPTN) and TANK-binding kinase 1 (TBK1) 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 @VARIANT$ missense mutation in trans, while case B carried a deletion of OPTN exons 13-15 (p.Gly538Glufs*27) and a loss-of-function mutation (p.Arg117*) in TBK1.

C9ORF72;10137

GRN;1577

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

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

0no label

The novel truncated variant in @GENE$ was not found in our "in-house" Saudi exome data (unpublished data from the Saudi Human Genome Project), 1000 Genome and gnomAD databases. The @VARIANT$ (p.R77C) variant in @GENE$ and @VARIANT$ (p.I80Gfs*13) mutation in S100A13 also segregated fully with ILD in Families 1B and 2.

S100A13;7523

S100A3;2223

c.229C>T;tmVar:c|SUB|C|229|T;HGVS:c.229C>T;VariantGroup:3;CorrespondingGene:6274;RS#:138355706;CA#:1116284

c.238-241delATTG;tmVar:c|DEL|238_241|ATTG;HGVS:c.238_241delATTG;VariantGroup:13;CorrespondingGene:6284

0no label

We observed that in 5 PCG cases heterozygous CYP1B1 mutations (p.A115P, p.E229 K, and @VARIANT$) co-occurred with heterozygous TEK mutations (@VARIANT$, 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.R368H;tmVar:p|SUB|R|368|H;HGVS:p.R368H;VariantGroup:1;CorrespondingGene:1545;RS#:79204362;CA#:119016

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

0no label

Three rare missense variants (@VARIANT$, L2118V, and E2003D) of the SPG11 gene were found. The high detection rate of missense variants of this gene is probably due to the large size of the coding region; therefore, we suggest that these SPG11 variants are unlikely to be deleterious. Variants in the @GENE$ gene are most commonly associated with autosomal recessive spastic paraplegia, although homozygous variants have been recently identified in juvenile ALS, and heterozygous missense variants in sALS. Variants in UBQLN2 have been shown to be a cause of dominant X-linked ALS. A previously reported (M392V,) and a novel variant (Q84H) were found in the @GENE$ 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.

SPG11;41614

UBQLN2;81830

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

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

0no label

DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = @GENE$, GJB3 = gap junction protein beta 3, GJB6 = gap junction protein beta 6, @GENE$ = microphthalmia-associated transcription factor. By screening other gap junction genes, another subject (SH175-389) carrying a single heterozygous @VARIANT$ in GJB2 allele harbored a single heterozygous @VARIANT$ mutant allele of GJB3 (NM_001005752) (SH175-389) with known pathogenicity (Figure 4D).

gap junction protein beta 2;2975

MITF;4892

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

p.A194T;tmVar:p|SUB|A|194|T;HGVS:p.A194T;VariantGroup:18;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, @VARIANT$ and c.5749G>T in @GENE$, c.238_239dupC in USH1C, and @VARIANT$ and c.10712C>T 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.3719G>A;tmVar:c|SUB|G|3719|A;HGVS:c.3719G>A;VariantGroup:87;CorrespondingGene:4647;RS#:542400234;CA#:5545997

c.2299delG;tmVar:c|DEL|2299|G;HGVS:c.2299delG;VariantGroup:190;CorrespondingGene:7399;RS#:80338903

0no label

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

MITF;4892

TYRO3;4585

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

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

0no label

@GENE$ Single Heterozygotes where DFNB1 was Excluded as a Final Molecular Diagnosis: A Fortuitously Detected GJB2 Mutation (Group I) There were three subjects (SH166-367, SH170-377, and SB175-334) with two recessive mutations, presumed to be pathogenic, in completely different deafness genes. One of the children with a heterozygous @VARIANT$ mutation (SH 166-367) was identified to carry a predominant founder mutation, p.R34X (c.100C>T) (rs121908073), and a novel variant, @VARIANT$ of @GENE$ (TMC1) (NM_138691), in a trans configuration (Table 1).

GJB2;2975

Transmembrane channel-like 1;23670

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

p.W482R;tmVar:p|SUB|W|482|R;HGVS:p.W482R;VariantGroup:0;CorrespondingGene:117531;RS#:754142954;CA#:5081956

11

Surprisingly, we identified two missense mutations in the proband: NM_001257180.2, exon10, c.1787A>G, @VARIANT$ in @GENE$ (Figure 1c) and NM_002609.4, exon3, c.317G>C, p.Arg106Pro, @VARIANT$ in @GENE$ (Figure 1d).

SLC20A2;68531

PDGFRB;1960

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

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

11

Moreover, a heterozygous p.Gly213Ser (@VARIANT$) mutation was detected in exon 3 of WNT10A, this leads to the substitution of Gly at residue 213 to Ser. Sequence analyses revealed that both mutant alleles were from his mother (Fig. 2D), who had a very mild phenotype of isolated tooth agenesis. His father did not have mutations in either of these genes. "S3" is a 14-year-old girl who had the typical clinical characteristics of HED: sparse hair, 26 missing permanent teeth, hypohidrosis, dry skin, and eczema on her body, but no plantar hyperkeratosis or nail abnormalities (Table 1). The heterozygous p.Arg156Cys (@VARIANT$) mutation was found in exon 3 of @GENE$, it results in the substitution of Arg at residue 156 to Cys. Additionally, the monoallelic p.Gly213Ser (c.637G>A) mutation was also detected in exon 3 of @GENE$, it results in the substitution of Gly at residue 213 to Ser.

EDA;1896

WNT10A;22525

c.637G>A;tmVar:c|SUB|G|637|A;HGVS:c.637G>A;VariantGroup:4;CorrespondingGene:80326;RS#:147680216;CA#:211313

c.466C>T;tmVar:c|SUB|C|466|T;HGVS:c.466C>T;VariantGroup:5;CorrespondingGene:1896;RS#:132630313;CA#:255655

0no label

Mutations in @GENE$ and @GENE$ in a patient with early-onset epileptic encephalopathy and respiratory depression Early infantile epileptic encephalopathy (EIEE) is a severe disorder associated with epilepsy, developmental delay and intellectual disability, and in some cases premature mortality. We report the case of a female infant with EIEE and strikingly suppressed respiratory dysfunction that led to death. Postmortem research evaluation revealed hypoplasia of the arcuate nucleus of the medulla, a candidate region for respiratory regulation. Genetic evaluation revealed heterozygous variants in the related genes NRXN1 (@VARIANT$, p.Arg896Trp) and NRXN2 (c.3176G>A, @VARIANT$), one inherited from the mother with family history of sudden infant death syndrome (SIDS) and one from the father with family history of febrile seizures.

NRXN1;21005

NRXN2;86984

c.2686C>T;tmVar:c|SUB|C|2686|T;HGVS:c.2686C>T;VariantGroup:1;CorrespondingGene:55777;RS#:796052777;CA#:316143

p.Arg1059Gln;tmVar:p|SUB|R|1059|Q;HGVS:p.R1059Q;VariantGroup:2;CorrespondingGene:9379;RS#:777033569;CA#:6078001

11

c, d) Sequence chromatograms indicating the wild-type, homozygous affected and heterozygous carrier forms of c) the C to T transition at position c.229 changing the arginine residue to cysteine at position 77 of the @GENE$ protein (@VARIANT$; p.R77C) and d) the @VARIANT$ (p.I80Gfs*13) in @GENE$. Mutation name is based on the full-length S100A3 (NM_002960) and S100A13 (NM_001024210) transcripts.

S100A3;2223

S100A13;7523

c.229C>T;tmVar:c|SUB|C|229|T;HGVS:c.229C>T;VariantGroup:3;CorrespondingGene:6274;RS#:138355706;CA#:1116284

c.238-241delATTG;tmVar:c|DEL|238_241|ATTG;HGVS:c.238_241delATTG;VariantGroup:13;CorrespondingGene:6284

11

CSS170323 carries a heterozygous missense variant c.630G>C(@VARIANT$) in MYOD1 and a heterozygous missense variant @VARIANT$(p.Ala64Thr) in @GENE$ (Table 2). CSS170323 presented with L2 hemivertebra and fused ribs (the right 11th rib and 12th rib). During mesoderm development, the expression of MEOX1 is increased by MYOD1 (Gianakopoulos et al., 2011), suggesting that these two variant potentially result in the cumulative perturbation of TBX6-mediated pathway. CSS161458 had a heterozygous splicing variant c.156-1G>C in RIPPLY1, as described above, and a heterozygous missense variant c.464G>T(p.Arg155Leu) in MYOD1 was also identified. Although no direct interaction between RIPPLY1 and MYOD1 has been reported, they may together dysregulate the TBX6 pathway given the deleterious nature of both variants (Table 2). DISCUSSION In this study, we performed exome sequencing on 584 patients with @GENE$ and without a molecular diagnosis.

MEOX1;3326

CS;56073

p.Met210Ile;tmVar:p|SUB|M|210|I;HGVS:p.M210I;VariantGroup:9;CorrespondingGene:4654;RS#:749634841;CA#:5906491

c.190G>A;tmVar:c|SUB|G|190|A;HGVS:c.190G>A;VariantGroup:5;CorrespondingGene:4222;RS#:373680176;CA#:8592682

0no label

In patient AVM359, one heterozygous VUS (c.589C>T [@VARIANT$]) in @GENE$ inherited from the mother and one likely pathogenic de novo heterozygous variant (c.1592G>A [@VARIANT$]) in SCUBE2 were identified (online supplementary table S2). @GENE$ functions as a coreceptor that enhances VEGF/VEGFR2 binding to stimulate VEGF signalling.

ENG;92

SCUBE2;36383

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

p.Cys531Tyr;tmVar:p|SUB|C|531|Y;HGVS:p.C531Y;VariantGroup:5;CorrespondingGene:57758;RS#:1212415588

0no label

Additionally, the monoallelic p.Gly213Ser (c.637G>A) mutation was also detected in exon 3 of @GENE$, it results in the substitution of Gly at residue 213 to Ser. Sequence analyses of her parents' genome revealed that the mutant alleles were from her mother (Fig. 2E), who only had microdontia of the upper lateral incisors. Her father did not carry mutations for either of these genes. "S4" is an 8-year-old boy who also had the typical characteristics and facial features of HED and was missing 28 permanent teeth, but he did not have plantar hyperkeratosis or nail abnormalities (Table 1). The @VARIANT$ (c.1045G>A) mutation in exon 9 of @GENE$ and heterozygous @VARIANT$ (c.511C>T) mutation in exon 3 of WNT10A were detected.

WNT10A;22525

EDA;1896

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

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

0no label

CONCLUSIONS We firstly identified the novel digenic heterozygous mutations by WES, @GENE$ p.307_308del and SCN5A p.R1865H, which resulted in LQTS with repeat syncope, torsades de pointes, ventricular fibrillation, and sinoatrial node dysfunction. KCNH2 p.307_308del 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. @GENE$ @VARIANT$ reduced the instability index of Nav1.5 protein and sodium current. All of these were closely related to young early-onset LQTS and sinoatrial node dysfunction. LIMITATIONS Our study was performed only in the statistical field on KCNH2 p.307_308del and SCN5A p.R1865H by WES and predisposing genes analyses. More cellular and animal research is needed to further investigate whether the coexisting interaction of KCNH2 @VARIANT$ and SCN5A p.R1865H increases the risk of the early-onset LQTS and sinoatrial node dysfunction.

KCNH2;201

SCN5A;22738

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

Two potential disease-causing mutations were identified: (d) ENAM: @VARIANT$/ p.Asn197Ilefs*81, which was previously reported to cause ADAI in multiple families (Hart, Hart, et al., 2003; Kang et al., 2009; Kida et al., 2002; Pavlic et al., 2007; Wright et al., 2011). (e) @GENE$ missense mutation c.1559G>A/@VARIANT$. All recruited affected family members (II:2, II:4, III:1, III:2, III:3, and III:5) were heterozygous for both of these (@GENE$ and LAMA3) mutations.

LAMA3;18279

ENAM;9698

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

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

0no label

Despite the absence of IgG detected in the supernatants of these cultures, no defect was observed in the generation of isotype switched IgG+ cells in II.2 (carrying both TNFRSF13B/@GENE$ @VARIANT$ and TCF3 @VARIANT$ mutations), compared to III.2, who has neither mutation. Her son, III.1, carrying the @GENE$ T168fsX191 mutation only, also generated a similar proportion of IgG+ switched cells.

TACI;49320

TCF3;2408

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

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

0no label

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

WNT10A;22525

EDA;1896

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

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

0no label

A total of 2 novel variants, @VARIANT$ and @VARIANT$, were located in a myeloperoxidase-like domain, the catalytic site of the enzyme (Fig. S3B). A total of 4 TSHR variants were found in 2 patients and were compound heterozygotes for 2 different TSHR mutations. The @GENE$ variant p.R450H was a recurrent inactivating mutation and p.C176R and p.K618 were novel. p.C176R is located in the leucine-rich repeat region of the extracellular domain and responsible for high-affinity hormone binding and p.R528S and p.K618* are located in the cytoplasmic loops (Fig. S3C). Patients with GIS had a higher tendency to be affected with mutations than patients with TD [25/32 (78%) vs. 6/11 (54%), Fig. 2]. Variants in TG, TSHR, DUOXA2, SLC5A5 and PROP1 genes were found exclusively in patients with GIS, and 1 variant in @GENE$ was found in patients with TD.

TSHR;315

TRHR;20707

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

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

0no label

Notably, proband P05 in family 05 harbored a de novo @GENE$ c.1664-2A>C variant. Since the FGFR1 @VARIANT$ 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 (DCC @VARIANT$) and a maternal variant (@GENE$ p. Arg1299Cys).

FGFR1;69065

CCDC88C;18903

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

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

0no label

Variants in all known WS candidate genes (@GENE$, EDNRB, MITF, PAX3, SOX10, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (@VARIANT$; p.Asn322fs) was identified in the @GENE$ 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.

EDN3;88

MITF;4892

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

M1, CYP1B1: @VARIANT$. M2, @GENE$: p.(E387K). M3, CYP1B1: p.(E173*). M4, PITX2: p.(P179T). M5, @GENE$: @VARIANT$. Arrows show the index cases.

CYP1B1;68035

PITX2;55454

p.(A179fs*18);tmVar:p|FS|A|179||18;HGVS:p.A179fsX18;VariantGroup:3;CorrespondingGene:1545;RS#:771076928

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

0no label

The novel Q84H variant affects the N-terminal ubiquitin-like domain of the @GENE$ 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 FUS protein. However, the two rare FUS variants (Y25C and P106L) that were detected in this study were located in the N-terminal "prion-like" Q/G/S/Y domain (amino acids 1-165) of the protein. Although the majority of FUS mutations linked to ALS are located in the extreme C-terminus of the protein, several studies show that N-terminal variants may also be damaging. In the @GENE$ gene, a known missense variant (@VARIANT$) and a novel non-frameshift deletion (K631del) were identified in our patient cohort. The patient (#90u) carrying the novel K631del deletion was a 37-year-old patient who also showed symptoms of frontotemporal dementia (FTD). This is in line with the data from previous studies; according to which, TBK1 is a causative gene of ALS-FTD. The NEK1 @VARIANT$ variant was also present in this patient.

ubiquilin-2;81830

TBK1;22742

I397T;tmVar:p|SUB|I|397|T;HGVS:p.I397T;VariantGroup:11;CorrespondingGene:29110;RS#:755069538;CA#:6669001

R261H;tmVar:p|SUB|R|261|H;HGVS:p.R261H;VariantGroup:2;CorrespondingGene:4750;RS#:200161705;CA#:203762

0no label

These results were confirmed for the His24Leu and @VARIANT$ variants when using the reporters for CYP11A1 and HSD17B3 (Figure 2B,C). In contrast, variant @VARIANT$ did not change the reporter activities of CYP11A1 and @GENE$ (Figure 2B,C). Expression of NR5A1 variants was assessed by Western blot in our cell model. As shown in Figure 2D, SF1 protein expression was similar for all studied NR5A1 variants. 3. Discussion Patients harboring NR5A1 variants manifest with extremely broad phenotypes, ranging from normal sex development to complete sex reversal. A lack of genotype-phenotype correlation has been widely questioned, and the hypothesis of additional genetic variations contributing to the complex and variable phenotype has been formulated. In this work, we report clinical and genetic data of seven 46,XY DSD patients with heterozygous NR5A1 variants with normal adrenal function. Using targeted gene panel analysis for sex development-related genes, we found a second likely disease causing/pathogenic variant in known DSD genes (STAR, @GENE$, ZFPM2) in three of six studied 46,XY DSD individuals, supporting the hypothesis of oligogenic disease.

HSD17B3;20089

AMH;68060

Cys30Ser;tmVar:p|SUB|C|30|S;HGVS:p.C30S;VariantGroup:5;CorrespondingGene:6662;RS#:1003847603;CA#:293780979

Cys301Tyr;tmVar:p|SUB|C|301|Y;HGVS:p.C301Y;VariantGroup:2;CorrespondingGene:6736

0no label

Interestingly, one FALS proband carried 3 variants, each of which has previously been reported as pathogenic: SOD1 p.G38R, @GENE$ p.P136L, and @GENE$ @VARIANT$. Nine apparently sporadic subjects had variants in multiple genes (Table 4), but only two were well-established ALS mutations: TARDBP p.G287S was found in combination with VAPB @VARIANT$ 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.

ANG;74385

DCTN1;3011

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

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

0no label

Moreover, the presence of other variants (KCNQ1-@VARIANT$, KCNH2-@VARIANT$, and KCNE1-p.G38S) could further enhance the effects of the mutant channels, thus resulting in incomplete penetrance and variable expressivity of the phenotype. On the contrary, in the mother, some other factors, including unknown genetic modifiers, could counteract the functional impairment of mutant channels, thereby protecting the asymptomatic @GENE$-p.C108Y mutation-positive subject from arrhythmia susceptibility. We cannot rule out the presence, in this family, of other polymorphisms that alter the function of different ion channels. For example, SNPs in the @GENE$ gene have been associated with greatly deranged QT intervals in healthy subjects and in LQTS patients.

KCNH2;201

NOS1AP;136252

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

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

0no label

Most had C9orf72 repeat expansion combined with another mutation (e.g. @GENE$ @VARIANT$ or @GENE$ A321V; Supplementary Table 6). A single control also had two mutations, P372R in ALS2 and @VARIANT$ in TARDBP.

VCP;5168

TARDBP;7221

R155H;tmVar:p|SUB|R|155|H;HGVS:p.R155H;VariantGroup:10;CorrespondingGene:7415;RS#:121909329;CA#:128983

A90V;tmVar:p|SUB|A|90|V;HGVS:p.A90V;VariantGroup:40;CorrespondingGene:23435;RS#:80356715;CA#:586343

0no label

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

CAPN3;52

RYR1;68069

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

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

0no label

In addition, we have confirmed that immunoreactive signal corresponding to the anti-ephrin-B2 antibody was colocalized with that to the anti-@GENE$ antibody in the inner ear (Supplementary Fig. 3g). These results suggest an important role of ephrin-B2 as an inducer of EphA2 endocytosis with the transmembrane binding partner, pendrin, while its effect is weaker than that of ephrin-A1. Aberrant regulation of pathogenic forms of pendrin via EphA2 Some pathogenic variants of pendrin are not affected by EphA2/ephrin-B2 regulation. a, b Immunoprecipitation of EphA2 with mutated pendrin. myc-pendrin A372V, L445W, Q446R, G672E were not co-immunoprecipitated with EphA2. Densitometric quantifications are shown (b). Mean +- SEM; one-way ANOVA with Bonferroni post hoc analyses; *p < 0.05; (n = 3). c, d Immunoprecipitation of EphA2 with mutated pendrin. Immunocomplex of myc-pendrin L117F, @VARIANT$ and @VARIANT$ was not affected. Densitometric quantifications are shown (d). Mean +- SEM; (n = 3). e, f Internalization of EphA2 and mutated @GENE$ triggered by ephrin-B2 stimulation.

EphA2;20929

pendrin;20132

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

Causative heterozygous mutations in @GENE$ (@VARIANT$/c.1145A > G) and @GENE$ (p.I1176L/c.3526A > C) were identified by whole exome sequencing performed on III-1 and IV-1. Sanger sequencing on available samples from consenting individuals was used for segregation analysis and confirmation of variants in individuals denoted by '+' and '#', respectively (b) Genotypes and phenotypes of various members in the family Individual GFI1 Blood MYO6 Hearing III-1 N382S (het) Neutropenia, monocytosis I1176L (het) Impaired III-3 WT Normal WT Normal IV-1 N382S (het) Neutropenia, monocytosis I1176L (het) Impaired IV-2 N382S (het) Neutropenia, monocytosis @VARIANT$ (het) Impaired

GFI1;3854

MYO6;56417

p.N382S;tmVar:p|SUB|N|382|S;HGVS:p.N382S;VariantGroup:1;CorrespondingGene:2672;RS#:28936381;CA#:119872

I1176L;tmVar:p|SUB|I|1176|L;HGVS:p.I1176L;VariantGroup:2;CorrespondingGene:4646;RS#:755922465;CA#:141060203

0no label

DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = @GENE$, GJB3 = @GENE$, GJB6 = gap junction protein beta 6, MITF = microphthalmia-associated transcription factor. By screening other gap junction genes, another subject (SH175-389) carrying a single heterozygous @VARIANT$ in GJB2 allele harbored a single heterozygous @VARIANT$ mutant allele of GJB3 (NM_001005752) (SH175-389) with known pathogenicity (Figure 4D).

gap junction protein beta 2;2975

gap junction protein beta 3;7338

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

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

0no label

Subsequently, genetic testing for the LQT1, LQT2, LQT3, LQT5, and @GENE$ genes identified a heterozygous @VARIANT$ (@VARIANT$) mutation of the @GENE$ gene (LQT2) and a heterozygous c.170T > C (p.Ile57Thr) unclassified variant (UV) of the KCNE2 gene (LQT6).

LQT6;71688

KCNH2;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

In this family, the @GENE$/TACI @VARIANT$ mutation appears to demonstrate a gene dosage effect on serum IgG levels. The brother who is homozygous (II.4) for the TNFRSF13B/TACI C104R mutation has the lowest IgG levels, and consistently generated fewer isotype switched and differentiated ASC in vitro, compared with other family members who are heterozygotes. The presence of concomitant mutations, such as the @GENE$ @VARIANT$ mutation seen in the proband, may explain the variable penetrance and expressivity of TNFRSF13B/TACI mutations in CVID.

TNFRSF13B;49320

TCF3;2408

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

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

11

Importantly, he had no coexistent mutations in CHD7, FGF8, FGFR1, PROK2, PROKR2, TAC3, TACR3, @GENE$, GNRHR, @GENE$, or KISS1R. The second patient (KS male C7) had a heterozygous c.757G>A (p.Ala253Thr) mutation (Figure 1; Table 1) affecting a completely conserved Ala253 residue (Figures S1-4). Using multiple sequence alignment (ESPRESSO), a protein model for the N-terminus was constructed. Both SSPIDER and INTERPROSURF analysis (Figure S4) suggest functional importance for Ala253; and SIFT predicts a deleterious effect for p.Ala253Thr. Although p.Ala253Thr did not alter splicing or quantitative mRNA expression (not shown), lymphoblast protein expression was consistently reduced by 50% in vitro. This @VARIANT$ mutation was identified in a male with sporadic KS, unilateral renal agenesis, and partial pubertal development. He also had a KAL1 deletion (c.488_490delGTT;@VARIANT$) (Table 1; Figure 1B) we characterized previously.

KAL1;55445

GNRH1;641

p.Ala253Thr;tmVar:p|SUB|A|253|T;HGVS:p.A253T;VariantGroup:3;CorrespondingGene:26012;RS#:142726563;CA#:5370407

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

0no label

Amino acid conservation analysis showed that seven of the 10 variants (@GENE$ p.G1122S, CELSR1 p.R769W, DVL3 p.R148Q, PTK7 p.P642R, SCRIB p.G1108E, SCRIB @VARIANT$ and @GENE$ @VARIANT$) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

CELSR1;7665

SCRIB;44228

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

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

0no label

We identified a novel compound heterozygous variant in @GENE$ c.1285dup (p.(Arg429Profs*72); a likely pathogenic novel variant affecting the conserved residue 354 in the functional domain of @GENE$ (c.1062C > G; p.(Asn354Lys)); a pathogenic new homozygous nucleotide change in BBS7 that leads to a @VARIANT$, c.763A > T, and a likely pathogenic homozygous substitution @VARIANT$ in BBS6, leading to the change p.(Cys412Phe).

BBS1;11641

BBS2;12122

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

c.1235G > T;tmVar:c|SUB|G|1235|T;HGVS:c.1235G>T;VariantGroup:15;CorrespondingGene:8195;RS#:1396840386

0no label

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

EDN3;88

PAX3;22494

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

These data also indicate that an alternate pathway is used for quality control of pro-@GENE$ when MAN1B1 alpha-mannosidase activity is reduced. DISCUSSION In this study, we describe identification and characterization of abnormalities in patients with homozygous mutations in two genes, a novel mutation in SEC23A, @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.

COL1A1;73874

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

0no label

Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (@VARIANT$/@VARIANT$, 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 @GENE$ have overlapping expression patterns in the cochlea.

Cx26;2975

Cx31;7338

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

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

0no label

Amino acid conservation analysis showed that seven of the 10 variants (CELSR1 p.G1122S, CELSR1 p.R769W, @GENE$ p.R148Q, PTK7 @VARIANT$, SCRIB @VARIANT$, SCRIB p.G644V and @GENE$ p.K618R) were located at highly conserved nucleotides in human, dog, mouse, rat, and zebrafish.

DVL3;20928

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

The p.Ile312Met (@VARIANT$) mutation in @GENE$ and heterozygous p.Arg171Cys (c.511C>T) mutation in WNT10A were detected. The coding sequence in exon 9 of EDA showed a C to G transition, which results in the substitution of @VARIANT$; also, the coding sequence in exon 3 of WNT10A showed a C to T transition at nucleotide 511, which results in the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome revealed that the mutant alleles were from his mother, who carried digenic heterozygous EDA and @GENE$ mutations at the same locus as that of N2 (Fig. 2B).

EDA;1896

WNT10A;22525

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

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

0no label

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. Variants in the @GENE$ gene result in impairment of retrograde axonal transport leading to progressive motor neuron degeneration in mice and have been described in a range of neurogenetic diseases, including Charcot-Marie-Tooth type 2O, spinal muscular atrophy, and hereditary spastic paraplegia. A few studies described heterozygous variants in the DYNC1H1 gene in fALS and sALS patients, suggesting its role in ALS. Based on our findings, we strengthen the potential link between DYNC1H1 variants and ALS. Given that there are genetic and symptomatic overlaps among many neurodegenerative diseases, it has been suggested that causative variants might play roles in multiple disorders. Two heterozygous variants (@VARIANT$ and R166C) were detected in the GBE1 gene.

MATR3;7830

DYNC1H1;1053

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

H398R;tmVar:p|SUB|H|398|R;HGVS:p.H398R;VariantGroup:18;CorrespondingGene:2632;RS#:755004170;CA#:2499769

0no label

Three variants of @GENE$ (NM_007123), @VARIANT$, C4870F, and G805A with unknown pathogenic potential were identified using TES (see Table S3, Supplemental Content, which illustrates variants or mutations of Usher syndrome type 2A (USH2A) and Ankyrin 1 (@GENE$) identified in SH 94-208). However, this subject showed no retinal abnormalities and only manifested severe SNHL with a mean hearing threshold of 75 dB HL, which was not compatible with type II Usher syndrome. Therefore, these variants of USH2A were excluded as causative deafness mutations. SH94-208 also showed the G1748S variant of ANK1 (NM_000037). Structural variations such as large genomic deletions involving ANK1 at chromosome 8p11.2p12 can lead to contiguous syndrome, with SNHL as one of the symptoms. However, the @VARIANT$ variant of ANK1 was a point mutation, not a structural variation (see Table S4, Supplemental Content, which illustrates depth of coverage of TES).

USH2A;66151

ANK1;55427

R5143C;tmVar:p|SUB|R|5143|C;HGVS:p.R5143C;VariantGroup:6;CorrespondingGene:7399;RS#:145771342;CA#:182576

G1748S;tmVar:p|SUB|G|1748|S;HGVS:p.G1748S;VariantGroup:19;CorrespondingGene:286;RS#:746486928;CA#:4727361

0no label

Analysis of the entire coding region of the @GENE$ gene revealed the presence of two different missense mutations (N166S and A194T) occurring in compound heterozygosity along with the 235delC and 299delAT of @GENE$ in 3 simplex families (235delC/@VARIANT$, @VARIANT$/A194T and 299delAT/A194T).

Cx31;7338

GJB2;2975

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

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

0no label

A new pathogenic variant in BBS2 affecting a conserved residue in the functional domain of BBsome protein (c.1062C > G; @VARIANT$) was found in compound heterozygous state in patient #1 together with the known pathogenic variant p.(Arg339*). A new homozygous nucleotide change in BBS7 that leads to a stop codon in position 255, c.763A > T, was identified in patient #3. BBS1, BBS2 and BBS7 share a partially overlapping portion of a functional domain, mutation of which results in the same disease phenotype. New pathogenic variants of BBS2 and @GENE$ lie in this portion. The variant in BBS7 is noteworthy, since very few Bardet-Biedl cases are reported in the literature. Indeed, only 35 variants in this gene are listed in the Human Gene Mutation Database (HGMD, https://portal.biobase-international.com/cgi-bin/portal/login.cgi). A homozygous substitution c.1235G > T in @GENE$, leading to p.(@VARIANT$), was also identified in an affected sibling of proband #12.

BBS7;12395

BBS6;10318

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

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 (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 @VARIANT$ mutation, and showed absence of only the left upper lateral incisor without other clinical abnormalities. No mutations in these genes were found in the father. Sequence analyses of EDA and @GENE$ genes.

EDA;1896

WNT10A;22525

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

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

GJB3;7338

GJB2;2975

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

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

0no label

Subsequently, genetic testing for the LQT1, LQT2, LQT3, LQT5, 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 @GENE$. Genetic screening revealed that both sons are not carrying the familial KCNH2 mutation.

LQT2;201

LQT6;71688

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

To investigate the effects of one candidate variant on mutant @GENE$ function, Western blotting and coimmunofluorescence were used to assess binding capacity, and leptomycin B exposure along with immunofluorescence was used to assess nuclear localization. Results: We describe a child who presented in infancy with combined pituitary hormone deficiencies and whose brain imaging demonstrated a small anterior pituitary, ectopic posterior pituitary, and a thin, interrupted stalk. WES demonstrated heterozygous missense mutations in two genes required for pituitary development, a known loss-of-function mutation in @GENE$ (c.253C>T;@VARIANT$) inherited from an unaffected mother, and a WDR11 (@VARIANT$;p.I436V) mutation inherited from an unaffected father.

WDR11;41229

PROKR2;16368

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

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

0no label

Previous studies suggested that heterozygous variants in the @GENE$ may be causative for adult-onset sALS. @GENE$ encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, @VARIANT$) were detected. The P11S variant affects the b isoform of the MATR3 protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms. Further evidence is required to elucidate the mechanism of pathogenicity of these alterations. We discovered several variants in ALS candidate and risk genes. In a patient with LMN-dominant ALS with slow progression, we found two novel variants (T2583I and @VARIANT$) in the DYNC1H1 gene.

ALS2;23264

MATR3;7830

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

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

0no label

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

SOX10;5055

TYRO3;4585

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

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

0no label

The proband's son (III.1) has inherited the TCF3 T168fsX191 mutation, but not the TNFRSF13B/@GENE$ @VARIANT$ mutation. The proband's clinically unaffected daughter (III.2) has not inherited either mutation. The @GENE$ @VARIANT$ mutation was absent in the proband's parents, indicating a de novo origin.

TACI;49320

TCF3;2408

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

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

0no label

In patient AVM226, we identified the compound heterozygous variants c.3775G>A (@VARIANT$) and @VARIANT$ (p.Gln989Leu) in @GENE$ (table 2). @GENE$ and DSCAM have similar neurodevelopmental functions and are essential for self-avoidance in the developing mouse retina.

DSCAM;74393

DSCAML1;79549

p.Val1259Ile;tmVar:p|SUB|V|1259|I;HGVS:p.V1259I;VariantGroup:5;CorrespondingGene:1826;RS#:1212415588

c.2966A>T;tmVar:c|SUB|A|2966|T;HGVS:c.2966A>T;VariantGroup:5;CorrespondingGene:83394;RS#:1212415588

0no label

While tagged versions of EphA2 @VARIANT$ and EphA2 T511M were effectively precipitated with Fc-fusion @GENE$ compared to EphA2 WT, Fc-fusion ephrin-B2 failed to pull down EphA2 G355R and T511M (Fig. 7a). Consistently, internalization of EphA2 G355R and EphA2 T511M with @GENE$ induced by ephrin-B2 but not ephrin-A1 was suppressed (Fig. 7b, c). On the other hand, the mutated forms of EphA2 did not affect their ability to bind to pendrin (Fig. 7d). Discussion Proper and polarized localization of transporters in cells is essential for their function. Various previously identified pendrin mutations cause pendrin cytoplasmic localization. A subset of these mutations, such as @VARIANT$, are known to cause mis-folding of the protein, leading to accumulation in the endoplasmic reticulum.

ephrin-A1;3262

pendrin;20132

G355R;tmVar:p|SUB|G|355|R;HGVS:p.G355R;VariantGroup:4;CorrespondingGene:1969;RS#:370923409;CA#:625329

H723R;tmVar:p|SUB|H|723|R;HGVS:p.H723R;VariantGroup:10;CorrespondingGene:5172;RS#:121908362;CA#:253307

0no label

The DNA sequencing chromatograms from the proband show two @GENE$ and one @GENE$ heterozygous mutations. While both LRP6 variants, p.(@VARIANT$) and p.(Asn1075Ser), were inherited from her father, the WNT10A mutation, @VARIANT$ was maternally derived.

LRP6;1747

WNT10A;22525

Ser127Thr;tmVar:p|SUB|S|127|T;HGVS:p.S127T;VariantGroup:1;CorrespondingGene:4040;RS#:17848270;CA#:6455897

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

11

Five anencephaly cases carried rare or novel CELSR1 missense variants, three of whom carried additional rare potentially damaging PCP variants: 01F377 (@GENE$ c.6362G>A and PRICKLE4 c.730C>G), 2F07 (CELSR1 c.8807C>T and DVL3 @VARIANT$), 618F05 (CELSR1 c.8282C>T and SCRIB @VARIANT$). One patient (f93-80) had a novel PTK7 missense variant (c.655A>G) with a rare @GENE$ missense variant (c.1892C>T).

CELSR1;7665

CELSR2;1078

c.1622C>T;tmVar:c|SUB|C|1622|T;HGVS:c.1622C>T;VariantGroup:5;CorrespondingGene:1857;RS#:1311053970

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

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 GJB2 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 A194T) occurring in compound heterozygosity along with the 235delC and 299delAT of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and @VARIANT$/A194T). In family A, a profoundly hearing impaired proband was found to be heterozygous for a novel A to G transition at nucleotide position 497 of GJB3, resulting in an asparagine into serine substitution in codon 166 (N166S) and for the 235delC of @GENE$ (Fig. 1b, d). Genotyping analysis revealed that the GJB2/235delC was inherited from the unaffected father and the @VARIANT$ of GJB3 was inherited from the normal hearing mother (Fig. 1a).

Cx31;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

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

TACI;49320

TCF3;2408

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

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

0no label