Human Genetics

, Volume 116, Issue 4, pp 292–299 | Cite as

Characterization of Usher syndrome type I gene mutations in an Usher syndrome patient population

  • Xiao Mei Ouyang
  • Denise Yan
  • Li Lin Du
  • J. Fielding. Hejtmancik
  • Samuel G. Jacobson
  • Walter E. Nance
  • An Ren Li
  • Simon Angeli
  • Muriel Kaiser
  • Valerie Newton
  • Steve D. M. Brown
  • Thomas Balkany
  • Xue Zhong Liu
Original Investigation

Abstract

Usher syndrome type I (USH1), the most severe form of this syndrome, is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. At least seven USH1 loci, USH1A-G, have been mapped to the chromosome regions 14q32, 11q13.5, 11p15, 10q21-q22, 21q21, 10q21-q22, and 17q24-25, respectively. Mutations in five genes, including MYO7A, USH1C, CDH23, PCDH15 and SANS, have been shown to be the cause of Usher syndrome type 1B, type 1C, type 1D, type 1F and type 1G, respectively. In the present study, we carried out a systematic mutation screening of these genes in USH1 patients from USA and from UK. We identified a total of 27 different mutations; of these, 19 are novel, including nine missense, two nonsense, four deletions, one insertion and three splicing defects. Approximatelly 35–39% of the observed mutations involved the USH1B and USH1D genes, followed by 11% for USH1F and 7% for USH1C in non-Acadian alleles and 7% for USH1G. Two of the 12 MYO7A mutations, R666X and IVS40-1G>T accounted for 38% of the mutations at that locus. A 193delC mutation accounted for 26% of CDH23 (USH1D) mutations, confirming its high frequency. The most common PCDH15 (USH1F) mutation in this study, 5601-5603delAAC, accounts for 33% of mutant alleles. Interestingly, a novel SANS mutation, W38X, was observed only in the USA cohort. The present study suggests that mutations in MYO7A and CDH23 are the two major components of causes for USH1, while PCDH15, USH1C, and SANS are less frequent causes.

Introduction

The Usher syndrome (USH) is an autosomal recessive disorder characterized by sensorineural hearing loss and retinitis pigmentosa (RP). It is estimated to account for approximately 50% of all individuals who are both deaf and blind (Boughman et al. 1983), 18% of those with RP and 3–6% of the congenitally deaf population (Vernon 1969). Usher syndrome is both clinically and genetically heterogeneous. Three clinical subtypes have been defined based on the onset of deafness and RP, and the presence or absence of vestibular dysfunction (Keats and Corey 1999). USH type I (USH1) is the most severe subtype, characterized by profound congenital sensorineural deafness, vestibular dysfunction and prepubertal onset RP. In USH2, the deafness is less severe and vestibular responses are normal. Finally, USH3 differs from USH1 and USH2 in that the hearing loss is progressive and RP and the vestibular dysfunction are more variable (Kimberling and Möller 1995; Smith et al. 1994). At least 11 loci have been mapped for Usher syndrome, seven of which have been cloned (Ahmed et al. 2003a). Mutations in five different genes can cause USH1: MYO7A (USH1B) encodes the actin-based motor protein myosin VIIa (Weil et al. 1995). USH1C encodes a PDZ (postsynaptic density, disc large, zonula occludens) domain-containing protein named harmonin (Verpy et al. 2000; Bitner-Glindzicz et al. 2000). Defects in the genes encoding two cadherin-related proteins, cadherin 23 and protocadherin 15, have been shown to cause USH1D (Bolz et al. 2001; Bork et al. 2001) and USH1F (Ahmed et al. 2001; Alagramam et al. 2001), respectively. The fifth USH1 gene, USH1G, codes for a scaffold protein and has been recently cloned. The deduced protein contains three ankyrin-like domains and a SAM (sterile alpha motif) domain motif, and its C-terminal tripeptide presents a class I PDZ-binding motif (Weil et al. 2003). Mutations in MYO7A have been reported to account for more than half of all type I cases (Astuto et al. 2000; Ahmed et al. 2003a), followed by approximately 30% linked to USH1D/USH1F (Astuto et al. 2000; Ahmed et al. 2003a).

In the present study, we report the results of a systematic screening for mutations in five cloned USH1 genes in a cohort of probands (59–130) with USH1 from the United States and United Kingdom.

Materials and methods

Subjects

Probands affected with USH1 from the United States and United Kingdom, as well as 100 control subjects, were sampled and studied. Mutation screening for the MYO7A (130 patients), USH1C (121 patients), CDH23 (74 patients), PCDH15 (103 patients) and SANS (59 patients) genes was performed. Patients screened for the USH1B, D, F and G genes had European ancestry. Ethnic background of the individuals screened for the Usher 1C gene has been previously reported (Ouyang et al. 2003). The clinical and family history was obtained on each proband, and complete physical examinations, including audiological and ophthalmologic examinations, were performed. Patients were identified as having USH1 according to the criteria recommended by the Usher Syndrome Consortium (Smith et al. 1994). Approval for human subjects for this study was obtained from institutional review board (IRB) at University of Miami. Informed consent was obtained for all participants.

DNA amplification and mutation screening

Blood samples were collected, and genomic DNA was extracted using a standard extraction method. Briefly, DNA samples from the probands were first screened for mutations in MYO7A, the negative cases were then screened for CDH23 and PCDH15 genes, followed by the analysis of SANS. A combination of single-strand conformation polymorphism (SSCP) analysis and direct sequencing was performed. MYO7A has 49 exons, with a coding region of 6,645 bp. A total of 52 pairs of primers covering the 49 exons of the MYO7A gene were used for screening (Levy et al. 1997). CDH23 is composed of 69 exons with a coding region of 10,062 bp. Polymerase chain reaction (PCR) amplification of CDH23 exons was performed using slightly modified versions of the primers described by Bolz et al. (2001). PCDH15 has 33 exons, with a coding region of 5,865 bp. Screening of the 33 exons of PCDH15 was performed using the primers reported by Alagramam et al. (2001) with some modifications. SANS has three exons, two of which are coding. The 1,380-bp open reading frame (ORF) is predicted to encode a 460-amino-acid protein. The two coding exons of the USH1G gene were amplified using primers described by Weil et al. (2003). PCR primers and annealing conditions for amplification are available on request.

Screening of mutations was performed by SSCP on PAGE-electrophoresis as previously described (Liu et al. 1997). DNA fragments displaying an abnormal SSCP pattern were directly sequenced on an automated sequencer (ABI-PRISM, model 3100). Samples were sequenced in both the forward and reverse directions to confirm the location of sequence change.

Results

Table 1 summarizes the mutations detected in patients with USH1 identified in this study. Table 2 lists the presumed nonpathogenic variants detected in these patients.
Table 1

Likely pathologic mutations in USH1 genes detected in USH1 patients

Base change

Codon change

Domain

Number of alleles

Genotype

Origin

A. MYO7A

 Missense

  47T>C

L16Sa

Motor domain

1

Compound heterozygote (with 521delC)

UKb

  73G>A

G25R

Motor domain

1

Compound heterozygote (with 809delC)

UKb

  494C>T

T165Ma

ATP binding domain

1

Heterozygote

UKb

  2266C>T

R756Wa

IQ motif

2

Heterozygote

UKb

  2904G>T

E968D

Post coiled coil domain

2

Homozygote

USAb

  5648G>A

R1883Qa

MyTH4 domain

1

Heterozygote

USAb

 Nonsense

  1996C>T

R666X

Motor domain

1

Compound heterozygote (with IVS27-G>C

UKb

   

4

Heterozygote

UKb

 Deletion

  2425delC

Q809FS

IQ3 motif

1

Compound heterozygote (with G25R)

UKb

   

1

Heterozygote

UKb

  1563delC

D521FS

Motor domain

1

Compound heterozygote (with L16S)

UKb

  2500delC

R834FSa

IQ4 motif

1

Heterozygote

USAb

 Splicing

  IVS27-1G>Ca

  

1

Compound heterozygote (withR666X)

UKb

   

2

Heterozygote

UKb

  IVS40-1G>Ta

  

1

Heterozygote

UKb

B. USH1C

 Insertion

  238-239insC

 

Amino terminus

2

Homozygote

UKc

  216G>A

 

Amino terminus

7

Compound heterozygote (with VNTR)

Acadia

  VNTR

  

2

Homozygous

Canada

   

7

Compound heterozygote (with G216A)

Acadia

 C. CDH23

 Missense

  1096G>A

A366Ta

CA4

3

Heterozygote

USAb

  3625A>G

T1209A

CA11/CA12

2

Homozygote

USAb

  4520G>A

R1507Qa

CA14

2

Homozygote

USAb

  9565C>T

R3189Wa

Cytoplasmic domain

1

Heterozygote

USAb

  9734C>T

S3245Fa

Cytoplasmic domain

2

Heterozygote

USAb

 Nonsense

  6319C>T

R2107X

CA20

2

Homozygote

USAb

 Deletion

  193delC

L65FS

CA1

5

Heterozygote

USAb

 Insertion

  6392-6393insA

T2131FSa

CA20

1

Compound heterozygote (with IVS47-1delCAG)

USAb

 Splicing

  IVS47-1delCAGa

 

CA20

1

Compound heterozygote (with 6392-6393insA)

USAb

 D. PCDH15

 Missense

  4024C>A

Q1342Ka

Pre-transmenbrane domain

1

Compound heterozygote (with5603delAAC)

USAb

 Nonsense

  2785C>T

R929Xa

CA9

1

Heterozygote

USAb

 Deletion

  16delT

Y6FSa

Signal peptide

1

Heterozygote

USAb

  996-999delGGAT

E332FSa

CA3

1

Heterozygote

UKb

  5603delAAC

T1867dela

Cytoplasmic domain

1

Compound heterozygote (with Q1342K)

USAb

   

1

Heterozygote

USAb

E. SANS

 Nonsense

  113G>A

W38X

Ankyrin repeat 1

4

Homozygote

USAb

aNovel changes in this study

bThe patients were of European ancestry

cPatient was from a family originated from Pakistan

Table 2

Polymorphisms detected in the USH1 genes

Genes

Nucleotide change

Codon change

Frequency

MYO7A

 

IVS12+8G>Aa

 

1/121

 

5715G>Aa

L1905L

2/121

 

6063G>Aa

L2021L

1/121

USH1C

 

IVS2-42insGGa

 

2/121

 

IVS3-16C>Ta

 

2/121

 

IVS16-18C>Ga

 

1/121

CDH23

 

IVS7+27G>Aa

 

1/76

 

IVS7+125G>Aa

 

1/76

 

1049C>T

F351F

1/76

 

2044G>Aa

Q683Q

3/76

 

3009C>T

S1003S

1/76

 

3664G>A

A1222T

5/76

 

4858G>A

V1620M

2/76

 

6197G>A

R2066R

1/76

 

6254G>Ta

G2085V

1/76

PCDH15

 

544A>Ga

G182G

2/113

 

IVS23+27T>Ga

 

1/113

 

IVS29+12T>Ca

 

1/113

 

IVS32-92delTa

 

1/113

 

IVS33-139insGTCa

 

3/113

 

IVS33+93T>Ca

 

1/113

 

IVS33+459A>Ga

 

1/113

 

5707A>Ga

Ile1903Val

1/113

aNovel changes in this study

MYO7A

Altogether, 12 likely pathogenic MYO7A sequence changes were identified, seven of which have not been previously reported. Four of six detected missense mutations are novel. A previously identified E968D mutation, located 32 amino acids downstream of the coiled coil region, was detected in a homozygous state in one individual. The L16S and G25R mutations were found in compound heterozygotes who also carried the 521delC and 809delC mutations, respectively. The remaining three missense mutations (T165M, R756W, R1883Q) were observed in heterozygotes in whom no second pathologic allele could be detected. The T165M change due to a C→T transition at nucleotide position 494 in exon 6 leads to an amino-acid substitution of a polar ambivalent residue, Thr, into a nonpolar internal residue, Met. This change occurs within the ATP binding site of the motor domain and therefore may destabilize the interactions within the ATP-binding site and affect ATPase activity. R756W, another C→T transition change affecting nucleotide 2266 in exon 19, results in the replacement of arginine by tryptophan. This change was detected in the neck region within the first IQ motif of MYO7A, involving the consensus amino acid of calmoduline-binding IQ motif, which therefore may impair the potential light-chains and/or calmodulin binding properties of the protein. The R1883Q mutation results from a G→A nucleotide substitution at position 5648 in exon 41, is located in the MyTH4 domain of the protein that is conserved between the human, mouse and rat myo7a. This change of a positively charged residue (arginine) into a smaller uncharged residue (glutamine) resides in the tail of the protein may well affect the membrane-binding function of the protein. A previously reported nonsense mutation (R666X) in exon 17 due to a C→ T transition that leads to a premature stop codon was identified. This mutation that would result in a truncated protein lacking approximately 90% of the predicted coding sequence was found in a compound heterozygous state with IVS27-G>C in one patient and in a heterozygous state in four patients who had no other detected pathologic mutation. Three different deletions were detected in the present study, one of which is novel. A previously reported mutation, 809delC (Levy et al. 1997), was found in a compound heterozygous state with G25R in one patient and in a heterozygous state in one case. The second deletion mutation consists of a deletion of a C at nucleotide position 1563, causing a frameshift starting at codon 521 and continuing for 7 amino acids and ending at a TGA stop codon. This mutation was observed in one patient in a compound heterozygous state with L16S mutation. Finally, a C deletion at nucleotide 2500 occurred in one affected individual in a heterozygous state. This mutation results in a frameshift at codon 834, followed by addition of 16 amino acids to the protein, before it terminates prematurely due to a premature stop codon. Two novel splice mutations in the MYO7A were detected in the present study. The first is a G→C transversion within the splice acceptor site of intron 27. This mutation was observed in a compound heterozygous state with R666X in one patient. Two additional probands were heterozygous for this mutation. The second splice mutation consists of a G→T transversion at position −1 in the intronic splice-acceptor region preceding exon 40. The mutation was detected in one affected individual in a heterozygous state. Those mutations identified within acceptor splice-sites are expected to result either in exon skipping or in frameshift due to cryptic splice-site usage. Both possibilities would seriously impair the proper construction of an effective mRNA. Mutations in MYO7A were identified in 17 out of 130 patients tested. In four of these, a mutation was found in both alleles. Of the 12 mutations detected in the MYO7A gene, the R666X and IVS40-1G>T mutations accounted for the greatest percentage of observed alleles (5/21=23.8%, 3/21=14.3%, respectively). So far, the majority of the reported MYO7A mutations are localized in the head region, this accumulation might be due to the higher screening effort concerning this region. Up until now, only two mutations in the neck domain (regulatory domain) have been reported (Adato et al. 1997; Levy et al. 1997); two additional mutations in that region are detected in the present study. The total number of pathologic mutations known to be located in the tail domain of the MYO7A protein (amino acids 857–2,215) involved in USH1B is 27. We found three additional mutations in the tail region of the protein that is known to vary dramatically, in both length and sequences, from one unconventional myosin to another and that is thought to reflect difference in membrane affinity (Titus 1997).

USH1C

We have previously reported two different mutations besides the nine-repeat VNTR in screening of USH1 patients in the USH1C gene (Ouyang et al. 2003). Data are summarized in Table 1. So far, only nine distinct USH1C mutations have been reported in USH1 patients (Ahmed et al. 2003a). Until recently, USH1C was believed to be a disease exclusive to the Acadian population of Louisiana (Astuto et al. 2000). However, after the gene was cloned, mutations were reported in individuals of different ethnic origins (Bitner-Glindzicz et al. 2000; Verpy et al. 2000; Zwaenepoel et al. 2001; Ahmed et al. 2002; Blaydon et al. 2003).

CDH23

A total of nine different CDH23 mutations were identified in a screening of 148 USH1 patient chromosomes in the present study. Six mutations occur within the CDH23-gene region encoding the ectodomains (EC or cadherin repeats which are involved in Ca2+-dependent cell adhesion); one lies within the linker region between two EC—repeat domains and two were found in the cytoplasmic domain. Four of the five missense mutations are novel. None of the mutations were found in 100 ethnically matched controls. The first is a G→A transition at the nucleotide position 1096 in exon 10 results in the substitution of an alanine for a threonine at codon 366 (A366T). Three individuals were heterozygous for this mutation. A previously reported mutation consisting of an A→G transition in exon 30 at codon 1209 located within the linker region between CA11 and CA12 (Astuto et al. 2002) was identified in one patient in a homozygous state. A replacement of an arginine residue by a glutamine (R1507Q), due to a G to A transition at position 4520 in exon 36, was another mutation detected in one patient in a homozygous state. Although the amino acids A366 and R1507 located in CA4 and CA14, respectively are not conserved within the EC domains, they are conserved between the human and mouse cdh23. To date, only three CDH23 mutations affecting the cytoplasmic part of the protein have been reported (Bork et al. 2001; Astuto et al. 2002) the majority of CDH23 mutations occur within the CDH23-gene region encoding the EC domain. We have identified two additional mutations in the cytoplasmic domain, one consisting of a C →T transition at nucleotide position 9565 in exon 67, affecting codon 3189 (R3189W). This mutation was detected in one patient in a heterozygous state. The second missense mutation located in the cytoplasmic region is a C →T transition in exon 68 that is expected to result in a S3245F substitution. This mutation was found to be carried by two individuals in a heterozygous state. The mutations in the cytoplasmic part might affect other functions, such as signal transduction pathways, rather than intercellular adhesion. The two newly identified cytoplasmic mutations reported here occur within the stretch of amino acids that have significant homology to a protein domain in the adaptor protein Ril, which functions as an internal PDZ-binding interface (PBI). Two CDH23 variants that differ by an insert of 105 bp have been previously described (Siemens et al. 2002). The 105 bp are encoded by an alternatively spliced exon (exon 68) that inserts 35 amino acids into the putative internal PBI of CDH23. The isoform containing exon 68 is believed to be preferentially expressed in inner ear sensory epithelia, which suggest that exon 68-encoded sequences regulate CDH23 function in the inner ear, marking this region as critical for the proper function of the protein. The R3189W change is located within the internal PBI domain of CDH23 (−68), whereas S3245 lies within the exon 68-encoded sequence specific to the inner ear. A previously reported nonsense mutation consisting of a C →T transition at nucleotide position 6319, affecting codon 2107, causing an arginine→stop (Bork et al. 2001), was found in a homozygous state in one individual. Two CDH23 mutations that created a frameshift leading to a subsequent premature stop codon were identified. The first mutation (193delC) consists of a deletion of a single C in a CCCCC string in exon 3 located in the first EC domain, causing a frameshift at codon 65 which continues for 48 amino acids and ends at a TGA stop codon. This mutation previously reported by Astuto et al. (2002) was found in a heterozygous state in five individuals. The second mutation is a one-base insertion of an A between nucleotide positions 6392 and 6393 in exon 47, resulting in a deduced TGA stop codon 34 amino acids downstream. As the mutant proteins in both cases lack the transmembrane domain, residual function is unlikely. Finally, a delCAG in the intronic splice-acceptor preceding exon 47 was observed in one individual in a compound heterozygous state with 6404insA. We have identified mutations in CDH23 in 15 probands out of the 76 tested. Four of these show biallelic mutations; in the remaining 61, only one mutation was detected. The previously reported 193delC is the most common mutation found in the CDH23 gene in the present study, accounting for ~26% (5/19) of the observed mutant alleles, followed by A366T, which represents ~16% (3/19). In relative abundance, 52% (10/19) of the CDH23 observed mutant alleles are missense mutations. None of the CDH23 missense mutations found in this study interfered with the evolutionarily conserved negatively charged motifs required for Ca2+ binding (LDRE, DXNDN and DXD), in contrast to what was seen for the missense mutations found in patients with DFNB12 (Astuto et al. 2002).

PCDH15

A total of five different previously unreported PCDH15 mutations were identified, including one missense mutation, one nonsense mutation and three deletions. The missense mutation found in the present study is a C→A transversion at nucleotide position 4024, leading to Q1342K substitution in exon 30. Q1342 is conserved between human and mouse protocadherin 15. This missense mutation located in the pre-transmembrane region was not found in 100 ethnically matched normal control subjects. The Q1342K was observed in a patient as a compound heterozygote with 5601–5603delAAC mutation (Table 1). A nonsense mutation (R929X) resulting from a C →T transition in exon 21 at nucleotide position 2785 was identified. This mutation, which would result in a truncated protein that lacks approximately 50% of the predicted coding sequence, is located in the ninth extracellular calcium-binding domain of protocadherin 15. This mutation was observed in one case as a heterozygote. Three different deletions were identified in our cohort. The first deletion consists of a single thymine deletion in a TTTT string in exon 2. This mutation causes a frameshift leading to an altered amino sequence from codon 6, followed by a premature stop at codon 11 in the predicted signal peptide sequence of PCDH15. This mutation was detected in a heterozygous state in one proband. The second type of deletion is a four-base deletion of a GGAT at nucleotide position 996–999 in exon 10, identified in a heterozygous state in one proband. This mutation causes a frameshift at codon 332, with a stop codon occurring 21 amino acids residues downstream. The third deletion is a three-base deletion of AAC (5601delAAC) in exon 33 at codon 1867, resulting in a deletion of a threonine residue at the mutation site. This three-base deletion located in the cytoplasmic domain was observed in a compound heterozygous state with Q1342K in one patient and in a heterozygous state in another proband. None of the previously reported mutations were observed in the present study. Mutations were identified in six out of the 103 patients screened for mutation in the PCDH15 gene. In only one of these was a mutation found in both alleles. In higher vertebrates, protocadherin genes appear to be involved in multiple roles, including neural development, neural circuit formation and formation of the synapse (Suzuki 2000). The function of protocadherins in the inner ear remains unclear. It may be a mediator of protein–protein interaction through the proline-rich regions in its cytoplasmic domain (Kay et al. 2000) and a similar functional role to that of cadherin 23 seems plausible.

SANS

Mutation screening of 118 USH1 patient chromosomes for the SANS gene resulted in the identification of a novel nonsense mutation. Two out of fifty nine probands (3.4%) from the United States were found to be homozygous for a G→A transition at nucleotide position 113, affecting codon 38 and resulting in the replacement of a tryptophan codon by a stop codon (W38X) at the mutation site. This nonsense mutation occurs in the first ankyrin domain of the SANS protein and would result in a truncated protein that lacks approximately 90% of the predicted coding sequence.

Table 2 lists the 23 polymorphisms identified in the four cloned USH1 genes in our typical USH1 cohort. Of these, 18 are novel. Only 3664G>A, 4858G>A, 6254G>T in CDH23 and 5707A>G in PCDH15 lead to a non-synonymous substitution in the translated protein. Twelve of the polymorphisms occur within the intronic regions. The polymorphisms detected in the present study were found to be relatively rare (one to six out of 160 alleles) among normal controls tested for their presence (data not shown).

Discussion

In the present study, altogether a total of 54 mutated alleles were identified in a screening of USH1 patients (59–130). In relative abundance and in almost equal proportion, 39% (21/54) and 35% (19/54) of observed mutant alleles are linked to loci USH1B and USH1D, respectively, followed by 7% (4/54) linked to USH1C (non-Acadian alleles) and 11% (6/54) to USH1F, with the 7% (4/54) to USH1G. Our present data thus provide evidence that mutations in MYO7A and CDH23 are common in USH1. However, our results do not support the view that USH1B is the most common subtype. The reported proportion of USH1 cases due to MYO7A defects is likely to be influenced not only by the number of patients analyzed but also by the ethnic background of the families. Another possible confounding factor to the measure of the proportion of USH1 cases due to a particular USH1 gene defect is the possible existence of mutations in regions of the gene that were not screened. Linkage and mutation analyzes so far indicated that 29–82% of USH1 cases were possibly the result of UHS1B alleles, depending on the population and number of exons screened (Larget-Piet et al. 1994; Weston et al. 1996; Adato et al. 1997; Janecke et al. 1999; Bharadwaj et al. 2000).

In this study, mutation screening of human MYO7A on 260 alleles of apparently unrelated patients with USH1 resulted in identification of 12 likely pathogenic alterations in the coding sequence and flanking intronic sequences of the gene. Given that only five mutations detected in that gene have been reported previously, it is likely that the majority of the MYO7A mutations arise as a single event, and that only a very small proportion of UHS1B alleles can be predicted in certain populations. Our data confirm that there is a wide range of heterogeneity in UHS1B. However, a relative homogeneity of UHS1B was found by Adato et al. (1997) from their screening of patients from Eastern Europe, Northern Africa, and the Middle East, which was not reflected in the present study nor in findings reported by Weston et al. (1996); the observation may reflect the relative isolation and marriage patterns among Jewish communities.

Altogether, mutations in UHS1 genes have been identified in 39 probands in the present study. In 28 individuals, only one mutation was detected, the remaining 11 showing biallelic mutations. The patients heterozygous for only one mutation could have a second mutation in the polyadenylation region, in the promoter region (which may lie within the first untranslated exon), or in enhancer elements at some distance from the coding exons, all of which were not screened. Additionally, although we have repeatedly screened all exons of the genes by SSCP and direct sequencing using different experimental conditions, no second aberration was detected. Clearly, we may have missed the second mutation in the amplicons analyzed, as neither SSCP nor direct sequencing, even combined, detects all mutations present. In addition, certain types of heterozygous mutations, e.g., exon-spanning deletions or inversions, escape detection if single-exon amplification is used.

Mutations in MYO7A, USH1C, CDH23 and PCDH15 cause both USH1 and nonsyndromic hearing loss. Clinically atypical cases that do not fit the three definitions of USH have also been reported with mutations in genes that usually cause typical type I (Liu et al. 1998). Previous studies have shown that there is a correlation between phenotype and type of mutations (Bolz et al. 2001; Bork et al. 2001; von Brederlow et al. 2002; Ahmed et al. 2003b). In the present study, we were not able to assign any specific phenotypic variation to the particular mutations. Currently there is consensus that the genotype does not always predict the clinical phenotype, which varies both within and between families carrying the same UHS1 gene mutations, implying the existence of other genetic and/or environmental factors that influence phenotype. Overall, the analysis of additional mutations in USH genes, combined with the study of mutated products and careful clinical descriptions should allow a more accurate estimation of possible direct phenotype–genotype correlation. Perhaps more importantly, these studies should also provide the basis for wide-scale segregation analysis for identification of “modifier genes” in the rest of the genome. From this knowledge, we should gain additional insight into the pathogenic processes involved in USH syndrome.

Notes

Acknowledgements

We thank the families for their participation in this study, which was supported in part by grants from the Foundation Fighting Blindness, NIH DC05575 (to X.Z.L.) and NIH EY-13385 (to S.G.J.). We thank Ms. S.B. Schwartz and Ms. E.E. Smilko for clinical coordination.

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Xiao Mei Ouyang
    • 1
  • Denise Yan
    • 1
  • Li Lin Du
    • 1
  • J. Fielding. Hejtmancik
    • 2
  • Samuel G. Jacobson
    • 3
  • Walter E. Nance
    • 4
  • An Ren Li
    • 2
  • Simon Angeli
    • 1
  • Muriel Kaiser
    • 2
  • Valerie Newton
    • 5
  • Steve D. M. Brown
    • 6
  • Thomas Balkany
    • 1
  • Xue Zhong Liu
    • 1
  1. 1.Department of Otolaryngology (D-48)University of MiamiMiamiUSA
  2. 2.National Eye Institute/NIHBethesdaUSA
  3. 3.Scheie Eye InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  4. 4.Department of Human GeneticsVirginia Commonwealth UniversityRichmondUSA
  5. 5.Center for AudiologyUniversity of ManchesterManchesterEngland
  6. 6.MRC Mouse Genome Centre and MRC Mammalian Genetics UnitEngland

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