Utilization of automated cilia analysis to characterize novel INPP5E variants in patients with non-syndromic retinitis pigmentosa.

INPP5E encodes inositol polyphosphate-5-phosphatase E, an enzyme involved in regulating the phosphatidylinositol (PIP) makeup of the primary cilium membrane. Pathogenic variants in INPP5E hence cause a variety of ciliopathies: genetic disorders caused by dysfunctional cilia. While the majority of these disorders are syndromic, such as the neuronal ciliopathy Joubert syndrome, in some cases patients will present with an isolated phenotype-most commonly non-syndromic retinitis pigmentosa (RP). Here, we report two novel variants in INPP5E identified in two patients with non-syndromic RP: patient 1 with compound heterozygous variants (c.1516C > T, p.(Q506*), and c.847G > A, p.(A283T)) and patient 2 with a homozygous variant (c.1073C > T, p.(P358L)). To determine whether these variants were causative for the phenotype in the patients, automated ciliary phenotyping of patient-derived dermal fibroblasts was performed for percent ciliation, cilium length, retrograde IFT trafficking, and INPP5E localization. In both patients, a decrease in ciliary length and loss of INPP5E localization in the primary cilia were seen. With these molecular findings, we can confirm functionally that the novel variants in INPP5E are causative for the RP phenotypes seen in both patients. Additionally, this study demonstrates the usefulness of utilizing ciliary phenotyping as an assistant in ciliopathy diagnosis and phenotyping.

The natural history and genotype-phenotype correlations of TMPRSS3 hearing loss: an international, multi-center, cohort analysis.

TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.

Chondrodysplasia, enchondromas and a chest deformity causing severe pulmonary morbidity in a boy with a PTHLH duplication: A case report.

Parathyroid hormone-like hormone (PTHLH) plays an important role in bone formation. Several skeletal dysplasias have been described that are associated with disruption of PTHLH functioning. Here we report on a new patient with a 898 Kb duplication on chromosome 12p11.22 including the PTHLH gene. The boy has multiple skeletal abnormalities including chondrodysplasia, lesions radiographically resembling enchondromas and posterior rib deformities leading to a severe chest deformity. Severe pulmonary symptoms were thought to be caused by limited mobility and secondary sputum evacuation problems due to the chest deformity. Imaging studies during follow-up revealed progression of the number of skeletal lesions over time. This case extends the phenotypic spectrum associated with copy number variation of PTHLH.

Further delineation of Malan syndrome.

Malan syndrome is an overgrowth disorder described in a limited number of individuals. We aim to delineate the entity by studying a large group of affected individuals. We gathered data on 45 affected individuals with a molecularly confirmed diagnosis through an international collaboration and compared data to the 35 previously reported individuals. Results indicate that height is > 2 SDS in infancy and childhood but in only half of affected adults. Cardinal facial characteristics include long, triangular face, macrocephaly, prominent forehead, everted lower lip, and prominent chin. Intellectual disability is universally present, behaviorally anxiety is characteristic. Malan syndrome is caused by deletions or point mutations of NFIX clustered mostly in exon 2. There is no genotype-phenotype correlation except for an increased risk for epilepsy with 19p13.2 microdeletions. Variants arose de novo, except in one family in which mother was mosaic. Variants causing Malan and Marshall-Smith syndrome can be discerned by differences in the site of stop codon formation. We conclude that Malan syndrome has a well recognizable phenotype that usually can be discerned easily from Marshall-Smith syndrome but rarely there is some overlap. Differentiation from Sotos and Weaver syndrome can be made by clinical evaluation only.

LONG-TERM FOLLOW-UP OF PATIENTS WITH RETINITIS PIGMENTOSA TYPE 12 CAUSED BY CRB1 MUTATIONS: A Severe Phenotype With Considerable Interindividual Variability.

PURPOSE: To examine the long-term clinical course and variability in a large pedigree segregating CRB1 type autosomal recessive retinitis pigmentosa. METHODS: An observational case study of 30 patients with CRB1 type autosomal recessive retinitis pigmentosa, homozygous for the CRB1 c.3122T > C; p.(Met1041Thr) mutation from a Dutch genetically isolated population in which the CRB1 gene was originally identified. The authors evaluated medical records, analyzed a questionnaire, and performed a comprehensive ophthalmic examination, including optical coherence tomography. RESULTS: Mean follow-up was 19 years (range 0-45 years, SD 15 years). With aging, patients showed progressive visual decline, deterioration of visual fields, increasing narrowing of the anterior chamber, increased prevalence of cataract, and an increase in the amount of intraretinal pigmentations. Fifty percent of patients had a visual acuity of ≤0.3 at Age 18 and of ≤0.1 at Age 35. Electroretinogram responses were severely reduced or absent already at a young age and optical coherence tomography showed increased retinal thickness with often cystoid maculopathy at young age, and thinning of the retina and disorientation of the photoreceptor layer in the late stages. The clinical course showed considerable interindividual variability, but intraindividual similarity between both eyes was the rule. CONCLUSION: The wide and variable clinical spectrum in patients with the same CRB1 mutation supports the hypothesis that the CRB1 type autosomal recessive retinitis pigmentosa-phenotype is modulated by other factors. The clinical variability will make it harder to evaluate the effect of (upcoming) therapies for retinitis pigmentosa, although because of the intraindividual similarity between both eyes, the contralateral eye can be used as an excellent internal control.

The diagnostic yield of whole-exome sequencing targeting a gene panel for hearing impairment in The Netherlands.

Hearing impairment (HI) is genetically heterogeneous which hampers genetic counseling and molecular diagnosis. Testing of several single HI-related genes is laborious and expensive. In this study, we evaluate the diagnostic utility of whole-exome sequencing (WES) targeting a panel of HI-related genes. Two hundred index patients, mostly of Dutch origin, with presumed hereditary HI underwent WES followed by targeted analysis of an HI gene panel of 120 genes. We found causative variants underlying the HI in 67 of 200 patients (33.5%). Eight of these patients have a large homozygous deletion involving STRC, OTOA or USH2A, which could only be identified by copy number variation detection. Variants of uncertain significance were found in 10 patients (5.0%). In the remaining 123 cases, no potentially causative variants were detected (61.5%). In our patient cohort, causative variants in GJB2, USH2A, MYO15A and STRC, and in MYO6 were the leading causes for autosomal recessive and dominant HI, respectively. Segregation analysis and functional analyses of variants of uncertain significance will probably further increase the diagnostic yield of WES.

ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report.

OBJECTIVE: Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. APPROACH AND RESULTS: Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations. CONCLUSIONS: Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.

Germline mutation of INI1/SMARCB1 in familial schwannomatosis.

Patients with schwannomatosis develop multiple schwannomas but no vestibular schwannomas diagnostic of neurofibromatosis type 2. We report an inactivating germline mutation in exon 1 of the tumor-suppressor gene INI1 in a father and daughter who both had schwannomatosis. Inactivation of the wild-type INI1 allele, by a second mutation in exon 5 or by clear loss, was found in two of four investigated schwannomas from these patients. All four schwannomas displayed complete loss of nuclear INI1 protein expression in part of the cells. Although the exact oncogenetic mechanism in these schwannomas remains to be elucidated, our findings suggest that INI1 is the predisposing gene in familial schwannomatosis.

ABCC6 and pseudoxanthoma elasticum.

ABCC6 belongs to the adenosine triphosphate-binding cassette (ABC) gene subfamily C. This protein family is involved in a large variety of physiological processes, such as signal transduction, protein secretion, drug and antibiotic resistance, and antigen presentation [Kool et al. (1999) 59:175-182; Borst and Elferink (2002) 71:537-592]. ABCC6 is primarily and highly expressed in the liver and kidney [Kool et al. (1999) 59:175-182; Bergen et al. (2000) 25:228-2231]. The precise physiological function and natural substrate(s) transported by ABCC6 are unknown, but the protein may be involved in active transport of intracellular compounds to the extracellular environment [Kool et al. (1999) 59:175-182] [Scheffer et al. (2002) 82:515-518]. Recently, it was shown that loss of function mutations in ABCC6 cause pseudoxanthoma elasticum (PXE) [Bergen et al. (2000) 25:228-2231; Le Saux et al. (2000) 25:223-227]. PXE is an autosomal recessively inherited multi-organ disorder [Goodman et al. (1963) 42:297-334; Lebwohl et al. (1994) 30:103-107]. PXE is primarily associated with the accumulation of mineralized and fragmented elastic fibers of the connective tissue in the skin [Neldner (1988) 6:1-159], Bruch's membrane in the retina [Hu et al. (2003) 48:424-438], and vessel walls [Kornet et al. (2004) 30:1041-1048]. PXE patients usually have skin lesions and breaks in Bruch's membrane of the retina (angioid streaks). Also, a variety of cardiovascular complications has been observed [Hu et al. (2003) 48:424-438]. Recently, a mouse model for PXE was created by targeted disruption of Abcc6 [Gorgels et al. (2005) 14:1763-1773; Klement et al. (2005) 25:8299-8310], which may be useful to elucidate the precise function of Abcc6 and to develop experimental therapies.

Does autosomal dominant pseudoxanthoma elasticum exist?

UNLABELLED: Pseudoxanthoma elasticum (PXE) is a progressive disorder of elastic fibers in skin, eyes, and arterial walls. It is caused by mutations in the ABCC6 gene. Most patients are sporadic cases. The majority of familial cases show autosomal recessive (AR) inheritance, but autosomal dominant (AD) inheritance has also been reported. We reviewed the literature on AD PXE and we studied in detail, both clinically and by DNA studies, a selection of potentially AD pedigrees from our patient population consisting of 59 probands and their family members. Individuals were considered to have definite PXE if they had two of the following three criteria: characteristic ophthalmologic signs, characteristic dermatologic signs, and a positive skin biopsy. In the literature we found only three families with definite PXE in two successive generations and no families with definite PXE in three or more generations. Our own data set comprised three putative AD families. Extensive DNA studies revealed a mutation in only one ABCC6 allele in the patients of these families. Only one of our families showed definite PXE in two generations. Linkage studies revealed that pseudodominance was unlikely in this family. In the other two families AD PXE could not be confirmed after extensive clinical examinations and application of our criteria, since definite PXE was not present in two or more generations. CONCLUSION: the inheritance pattern in PXE usually is AR. Part of the phenotype in family members of PXE patients might be due to expression in heterozygous carriers of an AR disease. AD inheritance in PXE may exist, but is both after careful literature study and in our patient material much rarer than previously thought.