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

CELSR1;7665

DVL3;20928

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

In Family A, there was digenic inheritance of two heterozygous variants: a novel variant in @GENE$ (c.3925G > A, p.Asp1309Asn) and a known DCM mutation in @GENE$ (c.2770G > A; @VARIANT$). The LAMA4 @VARIANT$ variant was predicted to be likely pathogenic according to international guidelines.

LAMA4;37604

MYH7;68044

p.Glu924Lys;tmVar:p|SUB|E|924|K;HGVS:p.E924K;VariantGroup:0;CorrespondingGene:4625;RS#:121913628;CA#:13034

p.Asp1309Asn;tmVar:p|SUB|D|1309|N;HGVS:p.D1309N;VariantGroup:1;CorrespondingGene:3910;RS#:782046057

0no label

The nucleotide sequence showed a T deletion at nucleotide 252 (c.252DelT) of the coding sequence in exon 1 of @GENE$; this leads to a frame shift from residue 84 and a premature @VARIANT$. Additionally, a monoallelic C to T transition at nucleotide 511 (@VARIANT$) of the coding sequence in exon 3 of WNT10A was detected, this leads to the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome showed that the mutant EDA allele was from his mother (Fig. 2C), however, we were unable to screen for @GENE$ mutations because of insufficient DNA.

EDA;1896

WNT10A;22525

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

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

0no label

Previously, Miura et al demonstrated a heterozygous c.1438A>G (@VARIANT$) @GENE$ variant in 1/65 IHH patients based upon sequence AY_255128 (now revised to c.1432A>G ;p.Thr478Ala from NP_056352). Since Thr478 was only partially conserved (Figure S1B) and no in vitro analysis was performed, its functional significance is unknown. No other IHH/KS genes were studied, so digenic disease cannot be excluded. One heterozygous NELF splice mutation (c.1159-14_22del) has been described. However, the only KS individual within the family also had a heterozygous @GENE$ mutation (@VARIANT$), suggesting digenic disease.

NELF;10648

FGFR1;69065

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

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

0no label

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

GJB3;7338

GJB2;2975

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

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

0no label

(A) In the @GENE$ exon 4 and exon 9, the arrows indicate the nucleotide substitution, c.475A > G and c.1051A > G, consisting, respectively, in the amino acid substitutions, S159G (A/G heterozygous patient and mother, A/A wild type father) and @VARIANT$; (B) in the @GENE$ exon 9 sequence, the @VARIANT$ substitution consisted in an amino acid substitution, K953E (A/G heterozygous patient and mother, A/A wild-type father).

IL10RA;1196

NOD2;11156

R351G;tmVar:p|SUB|R|351|G;HGVS:p.R351G;VariantGroup:0;CorrespondingGene:3587;RS#:8178561

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

11

In the present study, we found two variants: the @VARIANT$ variant in two patients and the A579T variant in one case, with both variants located within the coiled-coil domain (amino acid positions 331-906) of the protein, which is not in line with previous findings. Without additional functional evidence, the pathogenicity of these variants is uncertain. Three rare missense variants (R2034Q, L2118V, and E2003D) of the SPG11 gene were found. The high detection rate of missense variants of this gene is probably due to the large size of the coding region; therefore, we suggest that these 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 (@VARIANT$) were found in the @GENE$ gene.

SPG11;41614

UBQLN2;81830

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

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

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 @VARIANT$ of @GENE$ in 3 simplex families (235delC/N166S, 235delC/A194T and 299delAT/A194T). In family A, a profoundly hearing impaired proband was found to be heterozygous for a novel A to G transition at nucleotide position 497 of GJB3, resulting in an @VARIANT$ (N166S) and for the 235delC of GJB2 (Fig. 1b, d).

Cx31;7338

GJB2;2975

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

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

0no label

Three families were heterozygous for a previously reported single-nucleotide ENAM deletion (@VARIANT$/p.Asn197Ilefs*81). One of these families also harbored a heterozygous @GENE$ mutation (c.1559G>A/@VARIANT$) that cosegregated with both the AI phenotype and the @GENE$ mutation.

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

11

DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = gap junction protein beta 2, @GENE$ = gap junction protein beta 3, 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 p.V193E in GJB2 allele harbored a single heterozygous @VARIANT$ mutant allele of GJB3 (NM_001005752) (SH175-389) with known pathogenicity (Figure 4D). This 2-year-old female showed severe autosomal recessive SNHL with a mean hearing threshold of 87.5 dB HL. Single Heterozygous @GENE$ Mutant Allele with Unknown Contribution to SNHL in Our Cohort (Group III) A 39-year-old female subject (SH94-208) showed the p.T123N variant of GJB2. The pathogenic potential of the @VARIANT$ variant is controversial.

GJB3;7338

GJB2;2975

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

p.T123N;tmVar:p|SUB|T|123|N;HGVS:p.T123N;VariantGroup:5;CorrespondingGene:2706;RS#:111033188;CA#:134964

0no label

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

TCF3;2408

TACI;49320

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

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

0no label

Genotyping analysis revealed that the @GENE$/@VARIANT$ was inherited from the unaffected father and the N166S of GJB3 was inherited from the normal hearing mother (Fig. 1a). In families F and K, a heterozygous missense mutation of a @VARIANT$ of @GENE$ that causes A194T, was found in profoundly deaf probands, who were also heterozygous for GJB2/235delC (Fig. 1g, i) and GJB2/299-300delAT (Fig. 1l, n), respectively.

GJB2;2975

GJB3;7338

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

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

0no label

Variant analysis and incidental findings Of the 19 variants identified in this study, four were previously reported as pathogenic disease causing variants: 1) GBE1 R524Ter; 2) @GENE$ G407RfrTer14; 3) NUBPL IVS8DC; 4) @GENE$ @VARIANT$. Two additional variants, p. T4823 M in RYR1 and @VARIANT$ in CACNA1S, were also previously reported in association with Core myopathy and Malignant Hyperthermia Susceptibility (MHS), respectively.

PCCB;447

OAT;231

Y299Ter;tmVar:p|SUB|Y|299,ER|T;HGVS:p.Y299,ERT;VariantGroup:10;CorrespondingGene:4942

p. R498L;tmVar:p|SUB|R|498|L;HGVS:p.R498L;VariantGroup:1;CorrespondingGene:779;RS#:150590855;CA#:78268

0no label

Two novel variants were identified in @GENE$, including one frameshift mutation (c.2060_2060delG, p.C687LfsX34) and one missense mutation (@VARIANT$, p.G505D). A novel missense mutation was found in DUOXA2 (c.398G>A, p.R133H). Besides 28 rare nonpolymorphic variants, two polymorphic variants in @GENE$, p.H678R and @VARIANT$, were commonly identified with frequencies of 0.19 and 0.286, respectively, which were higher than those in the controls (0.19 versus 0.092, OR (odds ratio) = 2.327, P = 0.097; 0.286 versus 0.085, OR = 4.306, P = 0.001).

TG;2430

DUOX2;9689

c.1514G>A;tmVar:c|SUB|G|1514|A;HGVS:c.1514G>A;VariantGroup:10;CorrespondingGene:6528;RS#:867829370

p.S1067L;tmVar:p|SUB|S|1067|L;HGVS:p.S1067L;VariantGroup:4;CorrespondingGene:50506;RS#:269868;CA#:7537960

0no label

Importantly, one of the TBK1 missense changes (NM_013254.3:c.2086G>A; @VARIANT$; case C) was recently reported in two Swedish ALS patients and was shown to impair the binding of TBK1 to OPTN in vitro. The 5 variants with CADD_Phred scores below 20 are less likely to be pathogenic and were located outside the previously reported domains implicated in disease (@GENE$), previously seen in controls (PFN1), or when identified in a family, did not segregate with the disease (@GENE$). CNV analysis of the 21 neurodegenerative disease genes using Ingenuity Variant Analysis software further identified one patient with a partial deletion of OPTN (NM_001008211.1:@VARIANT$; p.Gly538Glufs27).

CSF1R;3817

FUS;2521

p.Glu696Lys;tmVar:p|SUB|E|696|K;HGVS:p.E696K;VariantGroup:6;CorrespondingGene:29110;RS#:748112833;CA#:203889

c.1243-740_1612+1292delins25;tmVar:c|INDEL|1243-740_1612+1292|25;HGVS:c.1243-740_1612+1292delins25;VariantGroup:37;CorrespondingGene:10133

0no label

Whole genome SNP genotyping, whole exome sequencing followed by Sanger validation of variants of interest identified a novel single nucleotide deletion mutation (@VARIANT$) in the @GENE$ gene. Moreover, a rare heterozygous, missense damaging variant (@VARIANT$; p.Val34Gly) in the C2orf74 has also been identified. The @GENE$ is an uncharacterized gene present in the linked region detected by DominantMapper.

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

In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (@VARIANT$), @GENE$ (@VARIANT$; p.L16V) and USH2A (c.9921T>G). Her father carries the mutations in MYO7A and USH2A without displaying symptoms of the disease, whilst her unaffected mother carries the mutation in USH1G. The mutations in MYO7A, USH1G and USH2A were not found in 666 control alleles. Of the four siblings, the affected girl is the only one who carries the mutations in @GENE$ and USH1G, and, all the more, the mutations in the three genes (Figure 2).

USH1G;56113

MYO7A;219

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

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

0no label

Results Cosegregating deleterious variants (GRCH37/hg19) in CACNA1A (NM_001127222.1: @VARIANT$, @VARIANT$), @GENE$ (NM_025232.3: c.109C>T, p.Arg37Trp), @GENE$ (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.

REEP4;11888

TOR2A;25260

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

The p.Ile312Met (c.936C>G) mutation in @GENE$ and heterozygous p.Arg171Cys (@VARIANT$) mutation in @GENE$ 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.

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

@GENE$ 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 (@VARIANT$) were identified in @GENE$ (table 2).

DSCAML1;79549

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

0no label

A single-nucleotide duplication (@VARIANT$/p.Pro133Alafs*13) replaced amino acids 133-1142 with a 12 amino acid (ATTKAAFEAAIT*) sequence, and a single-nucleotide deletion (c.2763delT/p.Asp921Glufs*32) replaced amino acids 921-1142 with 31 amino acids (ESSPQQASYQAKETAQRRGKAKTLLEMMCPR*). Three families were heterozygous for a previously reported single-nucleotide @GENE$ deletion (c.588+1delG/p.Asn197Ilefs*81). One of these families also harbored a heterozygous @GENE$ mutation (c.1559G>A/@VARIANT$) that cosegregated with both the AI phenotype and the ENAM mutation.

ENAM;9698

LAMA3;18279

c.395dupA;tmVar:c|DUP|395|A|;HGVS:c.395dupA;VariantGroup:18;CorrespondingGene:13801

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

0no label

Moreover, heterozygous missense variants in SNAI3 (c.607C>T; p.Arg203Cys) and @GENE$ (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients. Variant in SNAI3 (@VARIANT$; p.Arg203Cys) gene is rare in population and is probably damaging and deleterious as predicted by PolyPhen2 and SIFT, respectively. Variant in TYRO3 (c.1037T>A; @VARIANT$) gene is present in population databases with high frequency (0.22 MAF) and is benign and tolerated as predicted by PolyPhen2 and SIFT, respectively. Studies have shown that WNT pathway genes including LEF-1 may modulate the WS2 phenotype in WS2 cases with MITF mutation. Therefore, exome data was searched for variants in WNT pathway genes (LEF-1, @GENE$, APC, ZNRF3, LRP4, LRP5, LRP6, ROR1, ROR2, GSK3, CK1, APC, BCL9, and BCL9L) as well.

TYRO3;4585

RNF43;37742

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

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

0no label

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

MYO7A;219

USH2A;66151

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

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

0no label

Previous studies suggested that heterozygous variants in the @GENE$ may be causative for adult-onset sALS. MATR3 encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, @VARIANT$) were detected. The P11S variant affects the b isoform of the @GENE$ protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms. Further evidence is required to elucidate the mechanism of pathogenicity of these alterations. We discovered several variants in ALS candidate and risk genes. In a patient with LMN-dominant ALS with slow progression, we found two novel variants (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, @GENE$, MITF, PAX3, @GENE$, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (@VARIANT$; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; p.Arg203Cys) and TYRO3 (c.1037T>A; @VARIANT$) gene was identified in the exome data of both patients.

EDNRB;89

SOX10;5055

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

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; p.Cys163del of KAL1) and NELF/@GENE$ (c. 1160-13C>T of @GENE$ and @VARIANT$; @VARIANT$ of TACR3).

TACR3;824

NELF;10648

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

Protein structure analysis We performed protein structure analysis on the two @GENE$ mutations (p.R171C and @VARIANT$) and two novel @GENE$ mutations (@VARIANT$ and p.I312M) that were identified in this study.

WNT10A;22525

EDA;1896

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

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

0no label

We observed that in 5 PCG cases heterozygous CYP1B1 mutations (@VARIANT$, p.E229 K, and p.R368H) co-occurred with heterozygous @GENE$ mutations (p.E103D, p.I148T, @VARIANT$, and p.G743A) indicating a potential digenic inheritance (Fig. 1a). None of the normal controls carried both the heterozygous combinations of CYP1B1 and TEK mutations. The TEK Q214P and G743A alleles were absent in 1024 controls, whereas very low frequencies of heterozygous TEK E103D (0.005) and I148T (0.016) alleles were found in the control population (Table 1). A compound heterozygous TEK mutation (p.E103D and p.E300G) was also observed in 1 family (PCG38). However, the remaining 20 PCG cases harboring a single heterozygous TEK mutation did not carry any additional mutation in the other 35 adult and childhood glaucoma-associated genes (Supplementary Fig. 1; Supplementary Table 1). The co-occurrence of heterozygous TEK and @GENE$ mutations as seen in our PCG cases were not observed in additional sets of POAG, ARS, Aniridia, and Peter's Anomaly patients.

TEK;397

CYP1B1;68035

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

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

0no label

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

GJB2;2975

GJB6;4936

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

Whole-exome sequencing testing more than 50 genes known to cause myopathy revealed variants in the @GENE$ (@VARIANT$), RYR1 (rs143445685), CAPN3 (rs138172448), and @GENE$ (@VARIANT$) genes.

COL6A3;37917

DES;56469

rs144651558;tmVar:rs144651558;VariantGroup:6;CorrespondingGene:1293;RS#:144651558

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

0no label

Here, we found that pendrin A372V, L445W, @VARIANT$, and G672E did not bind to @GENE$. Given the fact that loss of EphA2 disturbs pendrin apical localization in vivo and cell surface presentation in vitro, the binding of @GENE$ with EphA2 might be critical for pendrin recruitment to the apical membrane in the inner ear and the thyroid. Thus, loss of the ability of pendrin to bind EphA2 may cause delocalization of pendrin from the plasma membrane. Additionally, we examined the binding ability of EphA2 to four membrane located forms of mutated pendrin. None of the mutants had impaired interaction with EphA2. However, @VARIANT$, which is known to have an intact transporter activity and membrane localization in cultured cells, showed compromised endocytosis after ephrin-B2 stimulation.

EphA2;20929

pendrin;20132

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

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

0no label

Notably, the patients carrying the p.T688A and p.I400V mutations, and three patients carrying the @VARIANT$ mutation also carry, in heterozygous state, p.Y217D, 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.V435I;tmVar:p|SUB|V|435|I;HGVS:p.V435I;VariantGroup:1;CorrespondingGene:10371;RS#:147436181;CA#:130481

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

0no label

Analysis of the proband's exome revealed four potential disease-causing mutations in FTA candidate genes: three heterozygous missense variants in @GENE$ (g.68531T>G, c.503T>G, @VARIANT$; g.112084C>G, c.2450C>G, p.Ser817Cys; g.146466A>G, c.4333A>G, p.Met1445Val) and one in @GENE$ (@VARIANT$, c.637G>A, p.Gly213Ser) (Figure 2A and Figure S2A,B).

LRP6;1747

WNT10A;22525

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

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

11

The latter individuals were also carriers of the @GENE$ nonsense mutation @VARIANT$. It should be noted that the mother and her twin sister were heterozygous for one of the @GENE$ missense mutation @VARIANT$ and one ABCC6 nonsense mutation p.R1141X, suggesting digenic inheritance of their cutaneous findings.

ABCC6;55559

GGCX;639

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

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

0no label

Notably, the patients carrying the p.T688A and p.I400V mutations, and three patients carrying the @VARIANT$ mutation also carry, in heterozygous state, p.Y217D, p.R268C (two patients), p.H70fsX5, and @VARIANT$ pathogenic mutations in KAL1, PROKR2, @GENE$, and @GENE$, respectively (Table 1), which further substantiates the digenic/oligogenic mode of inheritance of KS.

PROK2;9268

FGFR1;69065

p.V435I;tmVar:p|SUB|V|435|I;HGVS:p.V435I;VariantGroup:1;CorrespondingGene:10371;RS#:147436181;CA#:130481

p.G687N;tmVar:p|SUB|G|687|N;HGVS:p.G687N;VariantGroup:7;RS#:727505376(Expired)

0no label

Genetic evaluation revealed heterozygous variants in the related genes @GENE$ (@VARIANT$, @VARIANT$) and @GENE$ (c.3176G>A, p.Arg1059Gln), 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.Arg896Trp;tmVar:p|SUB|R|896|W;HGVS:p.R896W;VariantGroup:1;CorrespondingGene:9378;RS#:796052777;CA#:316143

0no label

A rare variant in @GENE$, c.428C>T; @VARIANT$, was detected in Case 2 and was classified as VUS. Finally, for Case 7 and her father, a previously reported ZFPM2/FOG2 (c.1632G>A; p.Met544Ile) pathogenic missense alteration was identified. No additional variants in other genes associated with testicular development and DSD were identified by our panel in Cases 3, 5 and 6. We were not able to further analyze Case 4 because of a lack of sample availability. 2.3. Transcription Activity and Protein Expression Testing of Novel NR5A1 Variants To study the impact of the three novel missense variants of NR5A1 on transactivation activity of regulated genes, HEK293 cells were co-transfected with WT or mutant NR5A1 expression vectors and three different promoter reporter constructs essential for steroid and sex hormone biosynthesis. All three novel @GENE$ variants had significantly reduced activity on the CYP17A1 reporter compared to WT (Figure 2A). These results were confirmed for the @VARIANT$ and Cys30Ser variants when using the reporters for CYP11A1 and HSD17B3 (Figure 2B,C).

AMH;68060

NR5A1;3638

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

His24Leu;tmVar:p|SUB|H|24|L;HGVS:p.H24L;VariantGroup:4;CorrespondingGene:6736;RS#:1262320780

0no label

We observed that isoproterenol could enhance the activity of LTCC in the HEK293T cells, which may be associated with the evocation of cAMP/protein kinase A pathways by the activation of the endogenous @GENE$ adrenoreceptors. In summary, we investigated an extremely rare large ERS family with a high incidence of nocturnal SCD, in which we found a pathogenic mutation in CACNA1C (@VARIANT$) with loss-of-function. The penetrance was also incomplete, which was modified by a gain-of-functional @GENE$-@VARIANT$ variant and sex.

beta2;4257

SCN5A;22738

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

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

0no label

However, @GENE$ p.R1865H showed no significant influence on the RNA structure (Figure 4c,d). The MFE of SCN5A p.R1865H mutation (-178.70 kcal/mol) was approximately similar to that of the wild type (-178.30 kcal/mol), which probably induced no obvious change in the centroid secondary structure. RNA secondary structural prediction. (a, b) Compared with wild-type @GENE$, the structure of KCNH2 p.307_308del affected the single-stranded RNA folding, resulting in a false regional double helix. The minimum free energy (MFE) of KCNH2 @VARIANT$ increased, which thus led to a reduction of structural stability. (c, d) SCN5A @VARIANT$ showed no significant influence on the RNA structure, and the MFE value of SCN5A p.R1865H mutation was approximately similar to that of the wild type Physical and chemical parameter prediction of protein Compared with the amino acids of wild-type KCNH2 (Table 3), KCNH2 p.307_308del showed a decreasing trend in molecular weight and increasing instability.

SCN5A;22738

KCNH2;201

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

0no label

Patient P0432 has a c.4030_4037delATGGCTGG (@VARIANT$) mutation in USH2A and a missense mutation in CDH23 (p.R1189W), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (c.46C>G; p.L16V) and USH2A (@VARIANT$). Her father carries the mutations in @GENE$ and @GENE$ without displaying symptoms of the disease, whilst her unaffected mother carries the mutation in USH1G.

MYO7A;219

USH2A;66151

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

c.9921T>G;tmVar:c|SUB|T|9921|G;HGVS:c.9921T>G;VariantGroup:115;CorrespondingGene:7399;RS#:1057519382

0no label

One subject tested homozygous for @GENE$ @VARIANT$, but we did not exclude the possibility of a deletion on one allele. The two additional subjects could each be compound heterozygotes comprised of a rare variant (p.C1554G or p.I2547T with 0.3% and 0.5% MAF in population database respectively) and a novel variant (p.R168Q or p.T14I respectively). The subject carrying p.I2547T and p.T14I was also heterozygous for @GENE$ @VARIANT$ which has previously been reported in a subject with SALS.

SETX;41003

TAF15;131088

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

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

0no label

After filtering for homozygous nonsynonymous single nucleotide variants (SNVs) within the linkage interval, that were either novel or had either low or unknown minor allele frequency in dbSNP, only three previously described variants, rs3795737 in @GENE$, @VARIANT$ in SETDB1 and @VARIANT$ in @GENE$, and one novel variant in S100A13, were identified.

ISG20L2;12814

S100A3;2223

rs143224912;tmVar:rs143224912;VariantGroup:2;CorrespondingGene:9869;RS#:143224912

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

0no label

In Family F, the @GENE$/@VARIANT$ was inherited from the unaffected father and the A194T of GJB3 was likely inherited from the normal hearing deceased mother (Fig. 1f). In Family K, genotyping analysis revealed that the father transmitted the @VARIANT$/@GENE$, while the mother is heterozygous for the GJB2/299-300delAT (Fig. 1k).

GJB2;2975

GJB3;7338

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

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

0no label

Moreover, given the complex association of LQTS mutants in the individuals of the family and the potential causative role of each mutation, and also based on in silico analysis and on evidence from previous studies, we focused on the in vitro functional characterization of the @GENE$-@VARIANT$ and KCNH2-p.C108Y variants. 2.3. Functional Consequences of the KCNQ1-p.R583H and KCNH2-p.C108Y Variants To investigate the functional consequences of KCNQ1-p.R583H and @GENE$-@VARIANT$, we performed whole cell patch clamp experiments in transiently transfected CHO-K1 cells.

KCNQ1;85014

KCNH2;201

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

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

0no label

Results In one family with four patients, we found evidence for the contribution of two co-inherited variants in two crucial genes expressed in the glomerular basement membrane (GBM); LAMA5-@VARIANT$ and COL4A5-@VARIANT$. Mutations in @GENE$ cause classical X-linked Alport Syndrome, while rare mutations in the @GENE$ have been reported in patients with focal segmental glomerulosclerosis.

COL4A5;133559

LAMA5;4060

p.Pro1243Leu;tmVar:p|SUB|P|1243|L;HGVS:p.P1243L;VariantGroup:2;CorrespondingGene:3911;RS#:756101090;CA#:9942875

p.Asp654Tyr;tmVar:p|SUB|D|654|Y;HGVS:p.D654Y;VariantGroup:4;CorrespondingGene:1287;RS#:1131692060

11

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; p.Asn322fs) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in SNAI3 (c.607C>T; @VARIANT$) and TYRO3 (@VARIANT$; p.Ile346Asn) gene was identified in the exome data of both patients.

EDN3;88

PAX3;22494

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

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

0no label

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 @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 WNT10A genes. (A) The EDA mutation c.769G>C and @GENE$ mutation c.511C>T were found in patient N1, who inherited the mutant allele from his mother.

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

In our study, @VARIANT$(p. Arg631*) and c.1267C > T(p. Arg423*) were the two reported variants, while @VARIANT$(p. Ser509fs) and c.1524del A(p. Ser509fs) were the two novel variants, which led to KS with small phallus, cryptorchidism, and obesity. Four kinds of KAl1 gene variants resulted in the termination of protein synthesis, the production of truncated protein, or the activation of nonsense-mediated mRNA degradation, which destroyed the integrity of the protein structure and led to the loss of protein function. @GENE$ (PROK2) is a protein that plays an important role in the development of olfactory nerve and GnRH neurons and the regulation of physiological rhythm through its receptor PROKR2. Meanwhile, KS patients present with homozygous, compound heterozygous, and heterozygous gene variants in the @GENE$ and PROKR2 genes, which can be passed down through autosomal dominant or oligogenic inheritance.

Prokineticin-2;9268

PROKR2;16368

c.1897C > T;tmVar:c|SUB|C|1897|T;HGVS:c.1897C>T;VariantGroup:9;CorrespondingGene:2260;RS#:121909642;CA#:130223

c.1525delA;tmVar:c|DEL|1525|A;HGVS:c.1525delA;VariantGroup:13;CorrespondingGene:3730

0no label

In AS patient IID27, the two mutations in @GENE$ and @GENE$ were inherited independently, likely indicating an in trans configuration. There is a splicing site mutation @VARIANT$ in COL4A5, inherited from her mother and a missense mutation c.4421C > T (p. (Thr1474Met)) inherited from her father (Figure 1a). In AS patient IID29, in addition to a glycine substitution (p. (@VARIANT$)) in COL4A3 in the heterozygous state, there was another heterozygous nonsense mutation c.5026C > T in COL4A4 genes.

COL4A5;133559

COL4A4;20071

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

Gly1119Asp;tmVar:p|SUB|G|1119|D;HGVS:p.G1119D;VariantGroup:21;CorrespondingGene:1285;RS#:764480728;CA#:2147204

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 c.824G>A; @VARIANT$ of TACR3).

NELF;10648

TACR3;824

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

Patient P0418 carries a nonsense mutation in USH2A (p.S5030X) and a missense mutation in MYO7A (@VARIANT$), but his brother, who is also clinically affected, does not carry the MYO7A mutation. Patient P0432 has a c.4030_4037delATGGCTGG (p.M1344fsX42) mutation in USH2A and a missense mutation in CDH23 (@VARIANT$), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (c.46C>G; p.L16V) 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 @GENE$. The mutations in @GENE$, USH1G and USH2A were not found in 666 control alleles.

USH1G;56113

MYO7A;219

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

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

0no label

However, @GENE$ p.R1865H showed no significant influence on the RNA structure (Figure 4c,d). The MFE of SCN5A p.R1865H mutation (-178.70 kcal/mol) was approximately similar to that of the wild type (-178.30 kcal/mol), which probably induced no obvious change in the centroid secondary structure. RNA secondary structural prediction. (a, b) Compared with wild-type @GENE$, the structure of KCNH2 p.307_308del affected the single-stranded RNA folding, resulting in a false regional double helix. The minimum free energy (MFE) of KCNH2 @VARIANT$ increased, which thus led to a reduction of structural stability. (c, d) SCN5A p.R1865H showed no significant influence on the RNA structure, and the MFE value of SCN5A @VARIANT$ mutation was approximately similar to that of the wild type Physical and chemical parameter prediction of protein Compared with the amino acids of wild-type KCNH2 (Table 3), KCNH2 p.307_308del showed a decreasing trend in molecular weight and increasing instability.

SCN5A;22738

KCNH2;201

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

0no label

We identified a novel compound heterozygous variant in BBS1 c.1285dup (p.(Arg429Profs*72); a likely pathogenic novel variant affecting the conserved residue 354 in the functional domain of @GENE$ (c.1062C > G; @VARIANT$); a pathogenic new homozygous nucleotide change in BBS7 that leads to a stop codon in position 255, c.763A > T, and a likely pathogenic homozygous substitution c.1235G > T in @GENE$, leading to the change p.(@VARIANT$).

BBS2;12122

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 ages of onset of the patients with the @GENE$ variants reported in this study were later than juvenile ALS onset, which generally manifests before 25 years of age. Previous studies suggested that heterozygous variants in the ALS2 may be causative for adult-onset sALS. MATR3 encodes three protein isoforms that have been described as nuclear-matrix and DNA/RNA binding proteins involved in transcription and stabilization of mRNA. In the present study, two novel heterozygous variants (P11S, @VARIANT$) were detected. The P11S variant affects the b isoform of the MATR3 protein (NM_001194956 and NP_001181885), contributing to splicing alteration of other isoforms. Further evidence is required to elucidate the mechanism of pathogenicity of these alterations. We discovered several variants in ALS candidate and risk genes. In a patient with LMN-dominant ALS with slow progression, we found two novel variants (T2583I and @VARIANT$) in the @GENE$ gene.

ALS2;23264

DYNC1H1;1053

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

Mutations of @GENE$ and SCN5A genes are closely related to LQTS. The mutations of KCNH2 @VARIANT$ and @GENE$ @VARIANT$ were found in the proband by WES and validated as positive by Sanger sequencing.

KCNH2;201

SCN5A;22738

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

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

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 Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric @GENE$/@GENE$ connexons.

Cx26;2975

Cx31;7338

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

Patient P0418 carries a nonsense mutation in USH2A (p.S5030X) and a missense mutation in MYO7A (p.K268R), but his brother, who is also clinically affected, does not carry the @GENE$ mutation. Patient P0432 has a c.4030_4037delATGGCTGG (@VARIANT$) mutation in USH2A and a missense mutation in CDH23 (p.R1189W), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G (c.46C>G; @VARIANT$) and USH2A (c.9921T>G). Her father carries the mutations in MYO7A and @GENE$ without displaying symptoms of the disease, whilst her unaffected mother carries the mutation in USH1G.

MYO7A;219

USH2A;66151

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

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

0no label

Although our present cohort did not carry homozygous changes in any of the known PCG genes, we reanalyzed our samples that harbored heterozygous mutations in any of these genes along with the @GENE$ mutations. We observed that in 5 PCG cases heterozygous @GENE$ mutations (@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).

TEK;397

CYP1B1;68035

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

Considering the facts that the loss-of-function mutations in FGFR1 were identified to act in concert with other gene defects and the @GENE$ @VARIANT$ variant was reported in a PSIS patient with an IHH-causative gene in a digenic manner, the possibility of oligogenic inheritance in family 05 cannot be ruled out. Six families harbored only one variant of IHH genes, but none had sufficient evidence to be identified as monogenic models. Among these variants, one was frameshift variant, immunoglobulin superfamily member 10 (@GENE$) @VARIANT$, and the rest were missense variants.

CCDC88C;18903

IGSF10;18712

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

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

0no label

Using SIFT and PolyPhen, the c.1777C > G variant in SLC9A6 was predicted to be damaging, but a different variant at the same amino acid, @VARIANT$ (p.Leu593Phe), was found in the ExAC database at a rate of 8.24 x 10-6. A male (ID041), unrelated to ID104, carried heterozygous missense variants c.1513G > A (@VARIANT$) in @GENE$ and c.353A > G (p.Asn118Ser) in @GENE$. He was seen at 7 years and 10 months and, at that time, was severely developmentally delayed in multiple domains (motor, cognitive, and language).

EHMT1;11698

MFSD8;115814

c.1777C > T;tmVar:c|SUB|C|1777|T;HGVS:c.1777C>T;VariantGroup:7;CorrespondingGene:10479;RS#:149360465;CA#:10524857

p.Gly505Ser;tmVar:p|SUB|G|505|S;HGVS:p.G505S;VariantGroup:4;CorrespondingGene:79813;RS#:757679895;CA#:5374656

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$. Additionally, a monoallelic @VARIANT$ (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.

WNT10A;22525

EDA;1896

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

(C) The sequence of the @VARIANT$ variant is well-conserved from humans to tunicates. (D) SH175-389 harbored a monoallelic p.V193E variant of @GENE$ and a monoallelic @VARIANT$ variant of @GENE$. DFNB1 = nonsyndromic hearing loss and deafness 1, GJB2 = gap junction protein beta 2, GJB3 = gap junction protein beta 3, GJB6 = gap junction protein beta 6, MITF = microphthalmia-associated transcription factor.

GJB2;2975

GJB3;7338

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

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

0no label

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 @GENE$ @VARIANT$), 2F07 (CELSR1 c.8807C>T and DVL3 c.1622C>T), 618F05 (CELSR1 c.8282C>T and @GENE$ @VARIANT$).

PRICKLE4;22752

SCRIB;44228

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

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

0no label

Variants in all known WS candidate genes (EDN3, @GENE$, MITF, PAX3, SOX10, SNAI2, and TYRO3) were searched and a novel rare heterozygous deletion mutation (c.965delA; @VARIANT$) was identified in the MITF gene in both patients. Moreover, heterozygous missense variants in @GENE$ (@VARIANT$; p.Arg203Cys) and TYRO3 (c.1037T>A; p.Ile346Asn) gene was identified in the exome data of both patients.

EDNRB;89

SNAI3;8500

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

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 Cx31 by sequencing. Analysis of the entire coding region of the @GENE$ gene revealed the presence of two different missense mutations (N166S and @VARIANT$) occurring in compound heterozygosity along with the 235delC and @VARIANT$ of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and 299delAT/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

We observed that in 5 PCG cases heterozygous CYP1B1 mutations (p.A115P, p.E229 K, and @VARIANT$) co-occurred with heterozygous @GENE$ 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.

TEK;397

CYP1B1;68035

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

The pathogenic potential of the @VARIANT$ variant is controversial. Three variants of @GENE$ (NM_007123), R5143C, @VARIANT$, and G805A with unknown pathogenic potential were identified using TES (see Table S3, Supplemental Content, which illustrates variants or mutations of Usher syndrome type 2A (USH2A) and @GENE$ (ANK1) identified in SH 94-208).

USH2A;66151

Ankyrin 1;55427

p.T123N;tmVar:p|SUB|T|123|N;HGVS:p.T123N;VariantGroup:5;CorrespondingGene:2706;RS#:111033188;CA#:134964

C4870F;tmVar:p|SUB|C|4870|F;HGVS:p.C4870F;VariantGroup:24;CorrespondingGene:7399

0no label

Most of the identified variants were heterozygous, except for two homozygous @GENE$ mutations (i.e., p.Lys530* and @VARIANT$) detected in two patients. Most variants were included in the databases or reported in previous studies, except for one heterozygous variant in @GENE$ (i.e., @VARIANT$) that was novel.

DUOX2;9689

TSHR;315

p.Arg1110Gln;tmVar:p|SUB|R|1110|Q;HGVS:p.R1110Q;VariantGroup:5;CorrespondingGene:50506;RS#:368488511;CA#:7537915

p. Ala579Val;tmVar:p|SUB|A|579|V;HGVS:p.A579V;VariantGroup:31;CorrespondingGene:7253

0no label

However, it was hard to determine whether the coexisting interactions of @GENE$ @VARIANT$ and SCN5A p.R1865H increased the risk of young and early-onset LQTS, or whether KCNH2 mutation was only associated with LQTS, while SCN5A mutation was only associated with sinoatrial node dysfunction. 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.

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

0no label

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

WNT10A;22525

EDA;1896

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

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

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

ephrin-B2;3019

pendrin;20132

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

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

0no label

Deleterious variants in HS1BP3 (NM_022460.3: c.94C>A, @VARIANT$) and @GENE$ (NM_004297.3: @VARIANT$, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP. Deleterious variants in DNAH17,TRPV4,CAPN11,@GENE$,UNC13B,SPTBN4,MYOD1, and MRPL15 were found in two or more independent pedigrees.

GNA14;68386

VPS13C;41188

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

DISCUSSION We present a Chinese family with PFBC in which the previously reported heterozygous mutation c.1787A>G (p.His596Arg) in SLC20A2 and the SNP (rs544478083) c.317G>C (p.Arg106Pro) in @GENE$ were identified. The proband's father with the @GENE$ @VARIANT$ (p.His596Arg) mutation showed obvious brain calcification but was clinically asymptomatic. The proband's mother with the PDGFRB c.317G>C (@VARIANT$) variant showed very slight calcification and was clinically asymptomatic.

PDGFRB;1960

SLC20A2;68531

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

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

0no label

Patient P0418 carries a nonsense mutation in USH2A (@VARIANT$) and a missense mutation in @GENE$ (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 @GENE$ and a missense mutation in CDH23 (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 (@VARIANT$; p.L16V) and USH2A (c.9921T>G).

MYO7A;219

USH2A;66151

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

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

0no label

The pathogenic potential of the @VARIANT$ variant is controversial. Three variants of USH2A (NM_007123), @VARIANT$, C4870F, and G805A with unknown pathogenic potential were identified using TES (see Table S3, Supplemental Content, which illustrates variants or mutations of Usher syndrome type 2A (@GENE$) and Ankyrin 1 (@GENE$) identified in SH 94-208).

USH2A;66151

ANK1;55427

p.T123N;tmVar:p|SUB|T|123|N;HGVS:p.T123N;VariantGroup:5;CorrespondingGene:2706;RS#:111033188;CA#:134964

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

0no label

The detected @VARIANT$ variant affects the nuclear localization signal 2 (amino acids 568-574) of the CCNF protein. A previously characterized pathogenic nonsense variant (@VARIANT$) and a rare missense alteration (R1499H) were detected in the ALS2 gene, both in heterozygous form. The alsin protein encoded by the @GENE$ gene is involved in endosome/membrane trafficking and fusion, cytoskeletal organization, and neuronal development/maintenance. Both homozygous and compound heterozygous variants in the ALS2 gene have been described as causative for juvenile ALS. The 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.

ALS2;23264

MATR3;7830

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

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

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 (@VARIANT$ 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).

GJB2;2975

Cx31;7338

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

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

11

The siblings we describe with the HNF1A P291fsinsC and @GENE$ @VARIANT$ mutations are the first cases of digenic transcription factor MODY where both mutations have previously been reported as being pathogenic. The @GENE$ P291fsinsC (@VARIANT$) mutation is the most common of all MODY mutations: it results in a frameshift and premature termination codon.

HNF4A;395

HNF1A;459

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

Results Genetic analyses detected two contributing variants located on different chromosomes in three unrelated probands: a heterozygous pathogenic mutation in @GENE$ (@VARIANT$, p.Pro392Leu) and a heterozygous variant in @GENE$ (@VARIANT$, p.Asn357Ser).

SQSTM1;31202

TIA1;20692

c.1175C>T;tmVar:c|SUB|C|1175|T;HGVS:c.1175C>T;VariantGroup:1;CorrespondingGene:8878;RS#:104893941;CA#:203866

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

11

Subsequently, genetic testing for the LQT1, LQT2, LQT3, @GENE$, and LQT6 genes identified a heterozygous c.3092_3096dup (p.Arg1033ValfsX26) mutation of the @GENE$ gene (LQT2) and a heterozygous @VARIANT$ (@VARIANT$) unclassified variant (UV) of the KCNE2 gene (LQT6).

LQT5;71688

KCNH2;201

c.170T > C;tmVar:c|SUB|T|170|C;HGVS:c.170T>C;VariantGroup:0;CorrespondingGene:3757;RS#:794728493;CA#:5221

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

0no label

25 The RYR3 (NM_001036: c.7812C > G, p.Asn2604Lys) and EBNA1BP2 (NM_001159936: c.1034A > T, @VARIANT$) variants were classified as likely benign and benign, respectively, while the TRIP6 (NM_003302: c.822G > C, p.Glu274Asp) and the CAPN9 (NM_006615: @VARIANT$, 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, @GENE$ 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 MSH6 and @GENE$, to cancer risk cannot be completely excluded.

TRIP6;37757

MUTYH;8156

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

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

0no label

Cases A and B carried nonsense mutations in @GENE$ (NM_001008211.1:c.703C>T; p.Gln235*), and TBK1 (NM_013254.3:c.349C>T; @VARIANT$) respectively; while the other 3 TBK1 mutations observed in cases C-E were missense changes. Importantly, one of the @GENE$ missense changes (NM_013254.3:c.2086G>A; @VARIANT$; case C) was recently reported in two Swedish ALS patients and was shown to impair the binding of TBK1 to OPTN in vitro.

OPTN;11085

TBK1;22742

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

p.Glu696Lys;tmVar:p|SUB|E|696|K;HGVS:p.E696K;VariantGroup:6;CorrespondingGene:29110;RS#:748112833;CA#:203889

0no label

Under the assumption of an autosomal recessive inheritance pattern, two variants were identified in @GENE$ (c.326_327insT, p.(Lys111Glnfs*27) and @VARIANT$, p.(Gln2373*)) (Table 1, S3 Fig.). Segregation analysis showed that they were both present on the maternal allele (Figs. 1C-D). By analyzing for the presence of variants in genes implicated in intellectual disability, hearing impairment and inherited retinal disease, a heterozygous stop mutation in @GENE$ (@VARIANT$, p.(Ser512*)), a gene previously shown to be involved in autosomal recessive RP.

RP1L1;105870

C2orf71;19792

c.7117C>T;tmVar:c|SUB|C|7117|T;HGVS:c.7117C>T;VariantGroup:3;CorrespondingGene:94137;RS#:759654067;CA#:4623074

c.1535C>A;tmVar:c|SUB|C|1535|A;HGVS:c.1535C>A;VariantGroup:1;CorrespondingGene:388939;RS#:1293811678

11

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

EDA;1896

WNT10A;22525

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

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

0no label

Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: @GENE$/KAL1 (c.757G>A; p.Ala253Thr of NELF and @VARIANT$; @VARIANT$ of @GENE$) and NELF/TACR3 (c. 1160-13C>T of NELF and c.824G>A; p.Trp275X of TACR3).

NELF;10648

KAL1;55445

c.488_490delGTT;tmVar:p|DEL|488_490|V;HGVS:p.488_490delV;VariantGroup:8;CorrespondingGene:26012

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

0no label

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

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

11

No mutations in SLC5A5, @GENE$, or @GENE$ gene exons were found. Most of the variants presented as heterozygous in patients. Only three variants were homozygous in three patients: (1) DUOX2: c.2779A>G (@VARIANT$) in one patient, (2) DUOX2:c.3329G>A (p.R1110Q) in one patient, and (3) DUOXA2: c.413dupA (@VARIANT$) in one patient.

TPO;461

IYD;12352

p.M927V;tmVar:p|SUB|M|927|V;HGVS:p.M927V;VariantGroup:27;CorrespondingGene:50506;RS#:755186335;CA#:7538155

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

0no label

All @GENE$ variants tested show a significant decrease in trans-activational activity with SOX9 and with @GENE$. Complete loss of trans-activational activity was noted for the proteins with missense mutations located in the LBD (@VARIANT$ and p.D364Y), the p.47_54 in-frame deletion, as well as both frame-shift mutations assayed (p.R89Gfs*17 and p.L209Cfs*87). The nonsense mutation SF1 (@VARIANT$;Y211*]) seemed to retain a low level of activity.

SF1;138518

SRY;48168

p.H310D;tmVar:p|SUB|H|310|D;HGVS:p.H310D;VariantGroup:4;CorrespondingGene:6662;RS#:780987236;CA#:8739053

p.[P210Q;tmVar:p|SUB|P|210|Q;HGVS:p.P210Q;VariantGroup:5;CorrespondingGene:2626;RS#:575307727;CA#:4630899

0no label

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

MYO15A;56504

TMC1;23670

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

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

0no label

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

EDA;1896

WNT10A;22525

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

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

0no label

The results showed that the two @GENE$ variants p.(H395N) and @VARIANT$ were associated with a 22%-28% reduced transactivation (Fig 5A). Only one of the two identified @GENE$ variants, @VARIANT$, could be cloned for functional evaluation.

FOXC2;21091

PITX2;55454

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

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

0no label

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

DCC;21081

CCDC88C;18903

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

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

0no label

Therefore, @GENE$ genotyping has not yet entered into clinical practice. On the basis of the data collected in this study, we may speculate that the presence of @GENE$-@VARIANT$, together with three KCNE1-@VARIANT$ alleles, could lead to an increased risk of developing cardiac arrhythmias due to the prolongation of the QT interval.

NOS1AP;136252

KCNH2;201

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

The hot spot variant c.533G > C (@VARIANT$) was found in six patients and located in the transmembrane domain of the protein, which could significantly reduce the release of ionized calcium and the signal activity. The FGFR1 gene is expressed in many tissues and plays an important role in the development of embryonic olfactory nerve and GnRH neurons mainly through the @GENE$/FGFR1 signalling pathway. FGFR1 signalling is essential for the migration, secretion, or survival of hypothalamic GnRH neurons and is widely expressed in the nervous and skeletal systems. The FGFR1 gene is inherited through an autosomal dominant mode. Loss of function can lead to both nIHH and KS, and more than 200 variants of the @GENE$ gene have been found in patients with IHH. The FGFR1 gene had the highest variant frequency, approximately 44% in our study, which was higher than that (10%) in the Caucasian population. Its variant can cause cleft lip and palate, short stature, and bone dysplasia. Among the 11 FGFR1 variants reported in our study, c.761G > A (p. Arg254Gln), @VARIANT$ (p. Arg78Cys), and c.2008G > A (p. Glu670Lys) were found to be pathogenic variants.

FGF;8822;8822

FGFR1;69065

p. Trp178Ser;tmVar:p|SUB|W|178|S;HGVS:p.W178S;VariantGroup:12;CorrespondingGene:128674;RS#:201835496;CA#:270917

c.232C > T;tmVar:c|SUB|C|232|T;HGVS:c.232C>T;VariantGroup:10;CorrespondingGene:2260

0no label

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

EDN3;88

EDNRB;89

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

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

GJB2;2975

Cx31;7338

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

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

0no label

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 @GENE$ gene revealed the presence of two different missense mutations (N166S and @VARIANT$) occurring in compound heterozygosity along with the @VARIANT$ and 299delAT of GJB2 in 3 simplex families (235delC/N166S, 235delC/A194T and 299delAT/A194T).

GJB2;2975

Cx31;7338

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

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 (Q84H) were found in the UBQLN2 gene. The novel @VARIANT$ variant affects the N-terminal ubiquitin-like domain of the ubiquilin-2 protein, which is involved in binding to proteasome subunits. FUS variants have been mostly detected in familial ALS cases that are localized within the C-terminus of the @GENE$ protein.

SPG11;41614

FUS;2521

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

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

0no label

Interestingly, one FALS proband carried 3 variants, each of which has previously been reported as pathogenic: @GENE$ p.G38R, ANG p.P136L, and DCTN1 @VARIANT$. Nine apparently sporadic subjects had variants in multiple genes (Table 4), but only two were well-established ALS mutations: @GENE$ @VARIANT$ was found in combination with VAPB p.M170I while a subject with juvenile-onset ALS carried a de novo FUS p.P525L mutation with a paternally-inherited intermediate-sized CAG expansion in ATXN2.

SOD1;392

TARDBP;7221

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

p.G287S;tmVar:p|SUB|G|287|S;HGVS:p.G287S;VariantGroup:0;CorrespondingGene:23435;RS#:80356719;CA#:586459

0no label

So, it is impossible to detect a mutation in a region which is not covered using this system (Case #9: @VARIANT$). Secondarily, the MPS system used in this study, is not effective for detecting homo-polymer regions, for example poly C stretch (Case #8: @VARIANT$). In addition, concerning pathogenecity of mutations identified, functional analysis will be necessary to draw the final conclusion in the future. In UK and US Caucasian USH1 patients, @GENE$ (MYO7A) has been reported as the most common USH1 genetic subtype, while @GENE$ (PCDH15) has been reported as the most common USH1 genetic subtype in North American Ashkenazi Jews.

USH1B;219

USH1F;23401

c.5821-2A>G;tmVar:c|SUB|A|5821-2|G;HGVS:c.5821-2A>G;VariantGroup:42;CorrespondingGene:64072

p.Lys542GlnfsX5;tmVar:p|FS|K|542|Q|5;HGVS:p.K542QfsX5;VariantGroup:6;CorrespondingGene:4647;RS#:782077721;CA#:6197531

0no label