Joubert Syndrome 2 (JBTS2) in Ashkenazi Jews Is Associated with a TMEM216 Mutation

Patients with Joubert syndrome 2 (JBTS2) suffer from a neurological disease manifested by psychomotor retardation, hypotonia, ataxia, nystagmus, and oculomotor apraxia and variably associated with dysmorphism, as well as retinal and renal involvement. Brain MRI results show cerebellar vermis hypoplasia and additional anomalies of the fourth ventricle, corpus callosum, and occipital cortex. The disease has previously been mapped to the centromeric region of chromosome 11. Using homozygosity mapping in 13 patients from eight Ashkenazi Jewish families, we identified a homozygous mutation, R12L, in the TMEM216 gene, in all affected individuals. Thirty individuals heterozygous for the mutation were detected among 2766 anonymous Ashkenazi Jews, indicating a carrier rate of 1:92. Given the small size of the TMEM216 gene relative to other JBTS genes, its sequence analysis is warranted in all JBTS patients, especially those who suffer from associated anomalies.     

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.     

C19orf12 mutation leads to a pallido-pyramidal syndrome

Pallido-pyramidal syndromes combine dystonia with or without parkinsonism and spasticity as part of a mixed neurodegenerative disorder. Several causative genes have been shown to lead to pallido-pyramidal syndromes, including FBXO7, ATP13A2, PLA2G6, PRKN and SPG11. Among these, ATP13A2 and PLA2G6 are inconsistently associated with brain iron deposition. Using homozygosity mapping and direct sequencing in a multiplex consanguineous Saudi Arabian family with a pallido-pyramidal syndrome, iron deposition and cerebellar atrophy, we identified a homozygous p.G53R mutation in C19orf12. Our findings add to the phenotypic spectrum associated with C19orf12 mutations.     

A mutation in COL9A1 causes multiple epiphyseal dysplasia: further evidence for locus heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.     

Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.     

Mutations in SNX14 Cause a Distinctive Autosomal-Recessive Cerebellar Ataxia and Intellectual Disability Syndrome

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.     

Mutations in the SLC3A1 Transporter Gene in Cystinuria

Cystinuria is an autosomal recessive disease characterized by the development of kidney stones. Guided by the identification of the SLC3A1 amino acid–transport gene on chromosome 2, we recently established genetic linkage of cystinuria to chromosome 2p in 17 families, without evidence for locus heterogeneity. Other authors have independently identified missense mutations in SLC3A1 in cystinuria patients. In this report we describe four additional cystinuria-associated mutations in this gene: a frameshift, a deletion, a transversion inducing a critical amino acid change, and a nonsense mutation. The latter stop codon was found in all of eight Ashkenazi Jewish carrier chromosomes examined. This report brings the number of disease-associated mutations in this gene to 10. We also assess the frequency of these mutations in our 17 cystinuria families.     

Rapid detection of the two common mutations in Ashkenazi Jewish patients with mucolipidosis type IV

Among Ashkenazi Jewish individuals with mucolipidosis IV (ML IV), two mutations in the ML IV gene, IVS3-1A --> G and delEX1-EX7, account for more than 95% of disease alleles. The reported method of genotyping for the delEX1-EX7 mutation involves a cumbersome multistep procedure. In the present study, a new simplified one-step procedure is described that detects this mutation in both patients and carriers. An improved procedure is also described for detection of the IVS3-1A --> G mutation. Using these improved procedures, we have characterized the ML IV mutant alleles in 27 patients and 95 of their relatives from 22 families, and in 123 unrelated and unaffected Ashkenazi Jewish controls. Of the 27 ML IV patients, 16 patients (59.3%) were found to be homozygous for the IVS3-1A --> G mutation and 1 patient (3.7%) homozygous for the delEX1-EX7 mutation. Additionally, 9 patients (33.3%) were compound heterozygotes for IVS3-1A --> G/delEX1-EX7. Among the 123 Ashkenazi Jewish controls, two individuals were identified as heteroallelic with one IVS3-1A --> G mutation (carrier frequency: approximately 1 in 61); none showed the delEX1-EX7 mutation. The modifications described here provide a more facile means of genotyping patients and carriers and expand the possibilities for screening at-risk populations.     

A missense mutation (L166P) in DJ-1, linked to familial Parkinson's disease, confers reduced protein stability and impairs homo-oligomerization

The identification of genetic mutations responsible for rare familial forms of Parkinson's disease (PD) have provided tremendous insight into the molecular pathogenesis of this disorder. Mutations in the DJ-1 gene cause autosomal recessive early onset PD in two European families. A Dutch kindred displays a large homozygous genomic deletion encompassing exons 1-5 of the DJ-1 gene, whereas an Italian kindred harbors a single homozygous L166P missense mutation. A homozygous M26I missense mutation was also recently reported in an Ashkenazi Jewish patient with early onset PD. Mutations in DJ-1 are predicted to be loss of function. The recent determination of the crystal structure of human DJ-1 demonstrates that it exists in a homo-dimeric form in vitro, whereas the L166P mutant exists only as a monomer. Here, we examine the in vivo effects of the pathogenic L166P and M26I mutations on the properties of DJ-1 in cell culture. We report that the L166P mutation confers markedly reduced protein stability to DJ-1, which results from enhanced degradation by the 20S/26S proteasome but not from a loss of mRNA expression. Furthermore, the L166P mutant protein exhibits an impaired ability to self-interact to form homo-oligomers. In contrast, the M26I mutation does not appear to adversely affect either protein stability, turnover by the proteasome, or the capacity of DJ-1 to form homo-oligomers. These properties of the L166P mutation may contribute to the loss of normal DJ-1 function and are likely to be the underlying cause of early onset PD in affected members of the Italian kindred.     

A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, RTEL1, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.     

Homozygous deletion of the very low density lipoprotein receptor gene causes autosomal recessive cerebellar hypoplasia with cerebral gyral simplification

An autosomal recessive syndrome of nonprogressive cerebellar ataxia and mental retardation is associated with inferior cerebellar hypoplasia and mild cerebral gyral simplification in the Hutterite population. An identity-by-descent mapping approach using eight patients from three interrelated Hutterite families localized the gene for this syndrome to chromosome region 9p24. Haplotype analysis identified familial and ancestral recombination events and refined the minimal region to a 2-Mb interval between markers D9S129 and D9S1871. A 199-kb homozygous deletion encompassing the entire very low density lipoprotein receptor (VLDLR) gene was present in all affected individuals. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. To our knowledge, this syndrome represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect.     

Anticipation in hereditary dentatorubral-pallidoluysian atrophy

Anticipation refers to the progressively earlier onset and increase in disease severity in successive generations. We studied four families with hereditary dentatorubral-pallidoluysian atrophy (DRPLA), a neurodegenerative disease, and anticipation was present in the mode of inheritance. In subsequent generations DRPLA shows an earlier onset and more severe as well as additional symptoms. Older onset patients suffer from cerebellar ataxia with or without dementia, whereas younger onset patients present as progressive myoclonus epilepsy syndrome, which consists of mental retardation, dementia, and cerebellar ataxia as well as epilepsy and myoclonus. Anticipation with paternal transmission was significantly greater than with maternal transmission.     

CA8 Mutations Cause a Novel Syndrome Characterized by Ataxia and Mild Mental Retardation with Predisposition to Quadrupedal Gait

We describe a consanguineous Iraqi family in which affected siblings had mild mental retardation and congenital ataxia characterized by quadrupedal gait. Genome-wide linkage analysis identified a 5.8 Mb interval on chromosome 8q with shared homozygosity among the affected persons. Sequencing of genes contained in the interval revealed a homozygous mutation, S100P, in carbonic anhydrase related protein 8 (CA8), which is highly expressed in cerebellar Purkinje cells and influences inositol triphosphate (ITP) binding to its receptor ITPR1 on the endoplasmatic reticulum and thereby modulates calcium signaling. We demonstrate that the mutation S100P is associated with proteasome-mediated degradation, and thus presumably represents a null mutation comparable to the Ca8 mutation underlying the previously described waddles mouse, which exhibits ataxia and appendicular dystonia. CA8 thus represents the third locus that has been associated with quadrupedal gait in humans, in addition to the VLDLR locus and a locus at chromosome 17p. Our findings underline the importance of ITP-mediated signaling in cerebellar function and provide suggestive evidence that congenital ataxia paired with cerebral dysfunction may, together with unknown contextual factors during development, predispose to quadrupedal gait in humans.     

Novel mutations of MYO15A associated with profound deafness in consanguineous families and moderately severe hearing loss in a patient with Smith-Magenis syndrome

Mutations in myosin XVA are responsible for the shaker 2 ( sh2) phenotype in mice and nonsyndromic autosomal recessive profound hearing loss DFNB3 on chromosome 17p11.2. We have ascertained seven families with profound congenital hearing loss from Pakistan and India with evidence of linkage to DFNB3 at 17p11.2. We report three novel homozygous mutations in MYO15A segregating in three of these families. In addition, one hemizygous missense mutation of MYO15A was found in one of eight Smith-Magenis syndrome (del(17)p11.2) patients from North America who had moderately severe sensorineural hearing loss.     

Offspring gender ratio and the rate of recurrent spontaneous miscarriages in jewish women at high risk for breast/ovarian cancer

BRCA1/BRCA2 germline mutations are associated with an increased breast/ovarian cancer risk. Offspring gender ratios may be skewed against male births in BRCA1 mutation carriers. In addition, the lack of viable homozygous BRCA1/BRCA2-mutation carriers implies that recurrent miscarriages may be associated with homozygous fetuses. Jewish Israeli high-risk women who were tested for being carriers of the predominant BRCA1/BRCA2 mutations in Jewish high-risk families were analyzed for the sex of offspring and the rate of spontaneous miscarriages. Overall, 817 women participated: 393 BRCA1/BRCA2-mutation carriers (229 with breast/ovarian cancer) and 424 high-risk noncarriers (208 with breast/ovarian cancer). No differences between the male-to-female offspring ratios of all study groups were noted. Among mutation carriers, the offspring male-to-female ratio was 0.97 (444 : 460), and among mutation carriers with cancer it was 0.92 (262 : 284). Similarly, no offspring gender skewing was noted among high-risk noncarriers, regardless of health status. The rates of three or more spontaneous miscarriages among participants with at least one live birth were 4.37% (15/343) among mutation carriers and 3% (12/401) among high-risk women (P = not significant). In conclusion, the offspring gender ratio is similar in high-risk Jewish families and in the general population. The issue of the rate of recurrent miscarriages in high-risk Jewish women is unresolved.     

Nonsense mutations in CABC1/ADCK3 cause progressive cerebellar ataxia and atrophy

Hereditary ataxias are genetic disorders characterized by uncoordinated gait and often poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs. Many ataxias are autosomal dominant, but autosomal recessive (AR) disease occurs as well. Homozygosity mapping in a consanguineous family with three affected children with progressive cerebellar ataxia and atrophy revealed a candidate locus on chromosome 1, containing the CABC1/ADCK3 (the chaperone, ABC1 activity of bc1 complex homologue) gene. CABC1/ADCK3 is the homologue of the yeast Coq8 gene, which is involved in the ubiquinone biosynthesis pathway. Mutation analysis of this gene showed a homozygous nonsense mutation (c.1042C > T, p.R348X). Eight additional patients with AR cerebellar ataxia and atrophy were screened for mutations in the CABC1/ADCK3 gene. One patient was compound heterozygous for the same c.1042C > T mutation and a second nonsense mutation (c.1136T > A, p.L379X). Both mutations created a premature stop codon, triggering nonsense mediated mRNA decay as the pathogenic mechanism. We found no evidence of a Dutch founder for the c.1042C > T mutation in AR ataxia. We report here the first nonsense mutations in CABC1 that most likely lead to complete absence of a functional CABC1 protein. Our results indicate that CABC1 is an important candidate for mutation analysis in progressive cerebellar ataxia and atrophy on MRI to identify those patients, who may benefit from CoQ10 treatment.     

Megalencephalic leukoencephalopathy with subcortical cysts; a founder effect in Israeli patients and a higher than expected carrier rate among Libyan Jews

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive inherited neurological disorder characterized by macrocephaly, deterioration in motor functions and cerebellar ataxia. In Israel the disease is found in an increased frequency among Libyan Jews. The disease is caused by mutations in the MLC1 gene, which encodes a putative CNS membrane transporter. We describe three novel mutations (p.G59E, p.P92S, and 134_136insC) in seven MLC families. One of these mutations, p.G59E, was found in the vast majority of MLC patients in Israel. Screening of 200 normal Libyan Jewish individuals for the p.G59E mutation, revealed a carrier rate of 1/40 compared with an expected carrier rate of 1/81. Several explanations could account for this difference the most likely one is an admixture of the Libyan Jewish population.     

Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A-->T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G-->A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein.     

Homozygosity mapping of Portuguese and Japanese forms of ataxia-oculomotor apraxia to 9p13, and evidence for genetic heterogeneity

Ataxia with oculomotor apraxia (AOA) is characterized by early-onset cerebellar ataxia, ocular apraxia, early areflexia, late peripheral neuropathy, slow progression, severe motor handicap, and absence of both telangiectasias and immunodeficiency. We studied 13 Portuguese families with AOA and found that the two largest families show linkage to 9p, with LOD scores of 4.13 and 3.82, respectively, at a recombination fraction of 0. These and three smaller families, all from northern Portugal, showed homozygosity and haplotype sharing over a 2-cM region on 9p13, demonstrating the existence of both a founding event and linkage to this locus, AOA1, in the five families. Three other families were excluded from this locus, demonstrating nonallelic heterogeneity in AOA. Early-onset cerebellar ataxia with hypoalbuminemia (EOCA-HA), so far described only in Japan, is characterized by marked cerebellar atrophy, peripheral neuropathy, mental retardation, and, occasionally, oculomotor apraxia. Two unrelated Japanese families with EOCA-HA were analyzed and appeared to show linkage to the AOA1 locus. Subsequently, hypoalbuminemia was found in all five Portuguese patients with AOA1 with a long disease duration, suggesting that AOA1 and EOCA-HA correspond to the same entity that accounts for a significant proportion of all recessive ataxias. The narrow localization of AOA1 should prompt the identification of the defective gene.