Electrostatic Compensation Restores Trafficking of the Autosomal Recessive Retinitis Pigmentosa E150K Opsin Mutant to the Plasma Membrane

Rhodopsin is the rod photoreceptor G protein-coupled receptor responsible for capturing light. Mutations in the gene encoding this protein can lead to a blinding disease called retinitis pigmentosa, which is inherited frequently in an autosomal dominant manner. The E150K opsin mutant associated with rarely occurring autosomal recessive retinitis pigmentosa localizes to trans-Golgi network membranes rather than to plasma membranes of rod photoreceptor cells. We investigated the molecular mechanisms underlying opsin retention in the Golgi apparatus. Electrostatic calculations reveal that the E150K mutant features an overall accumulation of positive charges between helices H-IV and H-II. Human E150K and several other closely related opsin mutants were then expressed in HEK-293 cells. Spectral characteristics and functional biochemistry of each mutant were analyzed after reconstitution with the cis-retinoid chromophore. UV-visible spectra and rhodopsin/transducin activation assays revealed only minor differences between the purified wild type control and rhodopsin mutants. However, partial restoration of the surface electrostatic charge in the compensatory R69E/E150K double mutant rescues the plasma membrane localization of opsin. These findings emphasize the fundamental importance of electrostatic interactions for appropriate membrane trafficking of opsin and advance our understanding of the pathophysiology of autosomal recessive retinitis pigmentosa due to the E150K mutation.     

Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Significance in Inherited Retinal Dystrophies

This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in RHO, c.937-2_944del, (ii) one rare homozygous mutation in C2orf71, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in ABCA4, c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of RPGR. The molecular findings for RHO and C2orf71 confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two ABCA4 mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the RPGR mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.     

Two novel mutations in the EYS gene are possible major causes of autosomal recessive retinitis pigmentosa in the Japanese population

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Leber's congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan.     

Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa

"Autosomal dominant retinitis pigmentosa" (adRP) refers to a genetically heterogeneous group of retinal dystrophies, in which 54% of all cases can be attributed to 17 disease loci. Here, we describe the localization and identification of the photoreceptor cell-specific nuclear receptor gene NR2E3 as a novel disease locus and gene for adRP. A heterozygous mutation c.166G-->A (p.Gly56Arg) was identified in the first zinc finger of NR2E3 in a large Belgian family affected with adRP. Overall, this missense mutation was found in 3 families affected with adRP among 87 unrelated families with potentially dominant retinal dystrophies (3.4%), of which 47 were affected with RP (6.4%). Interestingly, affected members of these families display a novel recognizable NR2E3-related clinical subtype of adRP. Other mutations of NR2E3 have previously been shown to cause autosomal recessive enhanced S-cone syndrome, a specific retinal phenotype. We propose a different pathogenetic mechanism for these distinct dominant and recessive phenotypes, which may be attributed to the dual key role of NR2E3 in the regulation of photoreceptor-specific genes during rod development and maintenance.     

Expression of Wild-Type Rp1 Protein in Rp1 Knock-in Mice Rescues the Retinal Degeneration Phenotype

Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4(th) and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3(rd) exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.     

A molecular case report of autosomal dominant retinitis pigmentosa: RP1/RHO sequence variants in a Turkish family

Retinitis pigmentosa (RP) is an inherited progressive retinal disease with a complex inheritance pattern affecting about 1 in 3,500 people worldwide. To date, a large number of sequence changes in the causal contributor genes of wide-spectrum heterogeneous RP were reported, including deletions, insertions, or substitutions that lead missense mutations or truncations. Here we present an association between the clinical presentations of adRP and sequence variants involving novel M216L mutation in the RHO gene together with nonsynonimous sequence changes R872H, N985Y, A1670T, S1691P, C2033Y, and synonimous Q1725Q with novel, N1521N, and T1733T SNPs in the RP1 gene of uncertain pathogenicity in a Turkish family with autosomal dominant retinitis pigmentosa.     

An autosomal dominant retinitis pigmentosa family with close linkage to D7S480 on 7q

Retinitis pigmentosa is the most prevalent inherited disorder of the retina. It can be autosomal dominant (adRP), autosomal recessive (arRP) or X-linked (XLRP). A form of adRP mapping to chromosome 7q was reported in a large Spanish pedigree. We have typed DNA from the members of another Spanish family for polymorphic markers from the known candidate genes. Positive lod scores were obtained only for the markers located on 7q31-35, giving a maximum lod score of 2.98 (3.01 by multipoint analysis) at = 0.00 for D7S480. A brief clinical evaluation is given.     

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.     

Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor.

We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.     

Mutation G827R in matriptase causing autosomal recessive ichthyosis with hypotrichosis yields an inactive protease

Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly(827) by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.     

Thermal Stability of Rhodopsin and Progression of Retinitis Pigmentosa: COMPARISON OF S186W AND D190N RHODOPSIN MUTANTS

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor''s active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin''s thermal stability and RP progression in patients.     

A new locus (RP31) for autosomal dominant retinitis pigmentosa maps to chromosome 9p

Retinitis pigmentosa (RP) is a debilitating disease of the retina affecting ∼1.5 million people worldwide. RP shows remarkable heterogeneity both clinically and genetically, with more than 40 genetic loci implicated, 12 of which account for the autosomal dominant form (adRP) of inheritance. We have recently identified a French Canadian family that presents with early onset adRP. After exclusion of all known loci for adRP, a genome-wide search established firm linkage with a marker from the short arm of chromosome 9 (LOD score of 6.3 at recombination fraction θ=0). The linked region is flanked by markers D9S285 and D9S1874, corresponding to a genetic distance of 31 cM, in the region 9p22-p13.     

A major locus for autosomal recessive retinitis pigmentosa on 6q, determined by homozygosity mapping of chromosomal regions that contain gamma-aminobutyric acid-receptor clusters.

Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, with extensive allelic and nonallelic genetic heterogeneity. Autosomal recessive RP (arRP) is the most common form of RP worldwide, with at least nine loci known and accountable for approximately 10%-15% of all cases. Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter in the CNS. Different GABA receptors are expressed in all retinal layers, and inhibition mediated by GABA receptors in the human retina could be related to RP. We have selected chromosomal regions containing genes that encode the different subunits of the GABA receptors, for homozygosity mapping in inbred families affected by arRP. We identify a new locus for arRP, on chromosome 6, between markers D6S257 and D6S1644. Our data suggest that 10%-20% of Spanish families affected by typical arRP could have linkage to this new locus. This region contains subunits GABRR1 and GABRR2 of the GABA-C receptor, which is the effector of lateral inhibition at the retina.     

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.

Methods

In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed.

Results

Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation.

Conclusions

This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.     

A Novel Missense SNRNP200 Mutation Associated with Autosomal Dominant Retinitis Pigmentosa in a Chinese Family

The SNRNP200 gene encodes hBrr2, a helicase essential for pre-mRNA splicing. Six mutations in SNRNP200 have recently been discovered to be associated with autosomal dominant retinitis pigmentosa (adRP). In this work, we analyzed a Chinese family with adRP and identified a novel missense mutation in SNRNP200. To identify the genetic defect in this family, exome of the proband was captured and sequencing analysis was performed to exclude known genetic defects and find possible pathogenic mutations. Subsequently, candidate mutations were validated in affected family members using Sanger sequencing. A novel missense mutation, c.2653C>G transition (p.Q885E), in exon 20 of SNRNP200 was identified. The mutation co-segregated with the disease phenotype over four generations and was absent in 100 normal unaffected individuals. This mutation occurs at highly conserved position in hBrr2 and is predicted to have a functional impact, suggesting that hBrr2-dependent small nuclear riboproteins (snRNPs) unwinding and spliceosome activation is important in the pathogenesis of some variants of RP.     

Exome Sequencing Identifies a Founder Frameshift Mutation in an Alternative Exon of USH1C as the Cause of Autosomal Recessive Retinitis Pigmentosa with Late-Onset Hearing Loss

We used a combined approach of homozygosity mapping and whole exome sequencing (WES) to search for the genetic cause of autosomal recessive retinitis pigmentosa (arRP) in families of Yemenite Jewish origin. Homozygosity mapping of two arRP Yemenite Jewish families revealed a few homozygous regions. A subsequent WES analysis of the two index cases revealed a shared homozygous novel nucleotide deletion (c.1220delG) leading to a frameshift (p.Gly407Glufs*56) in an alternative exon (#15) of USH1C. Screening of additional Yemenite Jewish patients revealed a total of 16 homozygous RP patients (with a carrier frequency of 0.008 in controls). Funduscopic and electroretinography findings were within the spectrum of typical RP. While other USH1C mutations usually cause Usher type I (including RP, vestibular dysfunction and congenital deafness), audiometric screening of 10 patients who are homozygous for c.1220delG revealed that patients under 40 years of age had normal hearing while older patients showed mild to severe high tone sensorineural hearing loss. This is the first report of a mutation in a known USH1 gene that causes late onset rather than congenital sensorineural hearing loss. The c.1220delG mutation of USH1C accounts for 23% of RP among Yemenite Jewish patients in our cohort.     

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca²⁺ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca²⁺ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca²⁺ activation and abolished luminal Ca²⁺ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [³H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.     

Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26)

Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Two novel mutations R653H and E230K in the Mediterranean fever gene associated with disease

Familial Mediterranean fever (FMF) is an autosomal recessive disorder caused by mutations in the Mediterranean fever gene (MEFV). We describe two novel missense mutations in MEFV, R653H and E230K. Both were found in compound heterozygosity with the mutation M694V in single Turkish patients with clinical syndromes characteristic for FMF. DNA sequencing and PCR-RFLP typing of the families confirmed the mutations and verified recessive modes of inheritance.