Joubert syndrome: a model for untangling recessive disorders with extreme genetic heterogeneity.

BACKGROUND: Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterised by hypotonia, ataxia, cognitive impairment, abnormal eye movements, respiratory control disturbances and a distinctive mid-hindbrain malformation. JS demonstrates substantial phenotypic variability and genetic heterogeneity. This study provides a comprehensive view of the current genetic basis, phenotypic range and gene-phenotype associations in JS. METHODS: We sequenced 27 JS-associated genes in 440 affected individuals (375 families) from a cohort of 532 individuals (440 families) with JS, using molecular inversion probe-based targeted capture and next-generation sequencing. Variant pathogenicity was defined using the Combined Annotation Dependent Depletion algorithm with an optimised score cut-off. RESULTS: We identified presumed causal variants in 62% of pedigrees, including the first B9D2 mutations associated with JS. 253 different mutations in 23 genes highlight the extreme genetic heterogeneity of JS. Phenotypic analysis revealed that only 34% of individuals have a 'pure JS' phenotype. Retinal disease is present in 30% of individuals, renal disease in 25%, coloboma in 17%, polydactyly in 15%, liver fibrosis in 14% and encephalocele in 8%. Loss of CEP290 function is associated with retinal dystrophy, while loss of TMEM67 function is associated with liver fibrosis and coloboma, but we observe no clear-cut distinction between JS subtypes. CONCLUSIONS: This work illustrates how combining advanced sequencing techniques with phenotypic data addresses extreme genetic heterogeneity to provide diagnostic and carrier testing, guide medical monitoring for progressive complications, facilitate interpretation of genome-wide sequencing results in individuals with a variety of phenotypes and enable gene-specific treatments in the future.

Dejerine-Sottas syndrome associated with point mutation in the peripheral myelin protein 22 (PMP22) gene.

Dejerine-Sottas syndrome is a hypertrophic, demyelinating neuropathy which appears to demonstrate autosomal recessive inheritance in most pedigrees. Clinical symptoms are similar but more severe than Charcot-Marie-Tooth disease type 1 (CMT1), of which the major subtype, CMT1A, results either from duplication of a 1.5-megabase DNA region in chromosome 17p11.2-p12 containing the myelin gene PMP22, or from PMP22 point mutation. Mutational analysis of the PMP22 coding region in two unrelated Dejerine-Sottas patients identified individual missense point mutations present in the heterozygous state. These findings suggest that Dejerine-Sottas syndrome can result from dominant point mutation alleles of PMP22.

Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene.

P0, a major structural protein of peripheral myelin, is a homophilic adhesion molecule and maps to chromosome 1q22-q23, in the region of the locus for Charcot-Marie-Tooth neuropathy type 1B (CMT1B). We have investigated P0 as a candidate gene in two pedigrees with CMT1B and found point mutations which are completely linked with the disease (Z = 5.5, theta = 0). The mutations, glutamate substitution for lysine 96 or aspartate 90, are located in the extracellular domain, which plays a significant role in myelin membrane adhesion. Individuals with CMT1B are heterozygous for the normal allele and the mutant allele. Our results indicate that P0 is a gene responsible for CMT1B.

Epilepsy and mental retardation limited to females: an X-linked dominant disorder with male sparing.

Several X-linked disorders affect females disproportionately or exclusively. These including focal dermal hypoplasia, oral-facial-digital syndrome type I (ref. 3) and epilepsy with bilateral periventricular heterotopias. X-linked dominant inheritance with male lethality is probably responsible for sex-limited expression of these disorders, as affected women have frequent spontaneous abortions and the sex ratio of their live offspring is often skewed. The same inheritance pattern has been proposed for Rett syndrome, Aicardi syndrome and microphthalmia with linear skin defects, but in these sporadic conditions, evidence of male lethality is lacking. We investigated an unusual family with epilepsy and mental retardation limited to females (EFMR, #121250 in ref. 9); this disorder is transmitted both by females and by completely unaffected carrier males. Assignment of the EFMR disease locus (EFMR) to the X chromosome indicates that selective involvement of females in X-linked disease may in some instances result from male sparing rather than male lethality.

A 2D crossover-based map of the human X chromosome as a model for map integration.

We have constructed a two-dimensional map of 243 markers on the X chromosome. The average distance between markers ordered by two recombinants is 5.4 centiMorgans (cM), which is reduced to 3.2 cM using a less stringent criterion of one recombinant. Map resolution is enhanced by replacing the usual reference marker format with a 2D format, and the two-recombinant rule is more conservative than the lod 3.0 criterion for order. Taken together, crossover mapping and the 2D format produces maps with greater reliability and higher resolution than maps constructed using currently accepted standards. This first high-density crossover-based map of an entire human chromosome provides a model for integrating physical and genetic maps.

The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.

IPEX is a fatal disorder characterized by immune dysregulation, polyendocrinopathy, enteropathy and X-linked inheritance (MIM 304930). We present genetic evidence that different mutations of the human gene FOXP3, the ortholog of the gene mutated in scurfy mice (Foxp3), causes IPEX syndrome. Recent linkage analysis studies mapped the gene mutated in IPEX to an interval of 17-20-cM at Xp11. 23-Xq13.3.

Microarray Analysis Reveals Overexpression of both Integral Membrane and Cytosolic Tight Junction Genes in Endometrial Cancer Cell Lines.

Deregulation of tight junction (TJ) proteins and the associated disruption of TJ function has been demonstrated to play a role in the development of endometrial cancer. In the current study, we have shown overexpression of claudin-3 and -4 mRNA (by RT-PCR) and protein (by immunoblotting) in a panel of 9 human endometrial cancer cell lines. To further expand our understanding of the complex role of TJ deregulation in endometrial cancer, we also investigated the expression of 84 TJ and TJ-associated genes (encoding the array of proteins that function within the TJ network from the membrane to nuclear signaling pathways) by microarray analysis. Consistent with the claudin-3 and -4 RT-PCR and immunoblot findings described above, we observed overexpression of the claudin-3 and -4 genes by microarray analysis. Further, we observed overexpression of an additional three genes in 8 of the 9 endometrial cancer cell lines: OCLN (occludin), F11R (JAM-A) and TJP3 (ZO-3). OCLN and F11R encode integral membrane proteins whereas TJP3 encodes a cytosolic scaffolding protein that indirectly links membrane TJ proteins to the actin cytoskeleton and cell signaling pathways. Our data suggest that the structural disruption of TJs coupled with the downstream deregulation of signaling pathways involved in cellular proliferation and migration may contribute to the development of endometrial cancer.

Mutations in 3 genes (MKS3, CC2D2A and RPGRIP1L) cause COACH syndrome (Joubert syndrome with congenital hepatic fibrosis).

OBJECTIVE: To identify genetic causes of COACH syndrome BACKGROUND: COACH syndrome is a rare autosomal recessive disorder characterised by Cerebellar vermis hypoplasia, Oligophrenia (developmental delay/mental retardation), Ataxia, Coloboma, and Hepatic fibrosis. The vermis hypoplasia falls in a spectrum of mid-hindbrain malformation called the molar tooth sign (MTS), making COACH a Joubert syndrome related disorder (JSRD). METHODS: In a cohort of 251 families with JSRD, 26 subjects in 23 families met criteria for COACH syndrome, defined as JSRD plus clinically apparent liver disease. Diagnostic criteria for JSRD were clinical findings (intellectual impairment, hypotonia, ataxia) plus supportive brain imaging findings (MTS or cerebellar vermis hypoplasia). MKS3/TMEM67 was sequenced in all subjects for whom DNA was available. In COACH subjects without MKS3 mutations, CC2D2A, RPGRIP1L and CEP290 were also sequenced. RESULTS: 19/23 families (83%) with COACH syndrome carried MKS3 mutations, compared to 2/209 (1%) with JSRD but no liver disease. Two other families with COACH carried CC2D2A mutations, one family carried RPGRIP1L mutations, and one lacked mutations in MKS3, CC2D2A, RPGRIP1L and CEP290. Liver biopsies from three subjects, each with mutations in one of the three genes, revealed changes within the congenital hepatic fibrosis/ductal plate malformation spectrum. In JSRD with and without liver disease, MKS3 mutations account for 21/232 families (9%). CONCLUSIONS: Mutations in MKS3 are responsible for the majority of COACH syndrome, with minor contributions from CC2D2A and RPGRIP1L; therefore, MKS3 should be the first gene tested in patients with JSRD plus liver disease and/or coloboma, followed by CC2D2A and RPGRIP1L.

Non-recurrent SEPT9 duplications cause hereditary neuralgic amyotrophy.

BACKGROUND: Genomic copy number variants have been shown to be responsible for multiple genetic diseases. Recently, a duplication in septin 9 (SEPT9) was shown to be causal for hereditary neuralgic amyotrophy (HNA), an episodic peripheral neuropathy with autosomal dominant inheritance. This duplication was identified in 12 pedigrees that all shared a common founder haplotype. METHODS AND RESULTS: Based on array comparative genomic hybridisation, we identified six additional heterogeneous tandem SEPT9 duplications in patients with HNA that did not possess the founder haplotype. Five of these novel duplications are intragenic and result in larger transcript and protein products, as demonstrated through reverse transcription-PCR and western blotting. One duplication spans the entire SEPT9 gene and does not generate aberrant transcripts and proteins. The breakpoints of all the duplications are unique and contain regions of microhomology ranging from 2 to 9 bp in size. The duplicated regions contain a conserved 645 bp exon within SEPT9 in which HNA-linked missense mutations have been previously identified, suggesting that the region encoded by this exon is important to the pathogenesis of HNA. CONCLUSIONS: Together with the previously identified founder duplication, a total of seven heterogeneous SEPT9 duplications have been identified in this study as a causative factor of HNA. These duplications account for one third of the patients in our cohort, suggesting that duplications of various sizes within the SEPT9 gene are a common cause of HNA.

Connexin mutations in X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMTX) is a form of hereditary neuropathy with demyelination. Recently, this disorder was mapped to chromosome Xq13.1. The gene for the gap junction protein connexin32 is located in the same chromosomal segment, which led to its consideration as a candidate gene for CMTX. With the use of Northern (RNA) blot and immunohistochemistry technique, it was found that connexin32 is normally expressed in myelinated peripheral nerve. Direct sequencing of the connexin32 gene showed seven different mutations in affected persons from eight CMTX families. These findings, a demonstration of inherited defects in a gap junction protein, suggest that connexin32 plays an important role in peripheral nerve.

Dysmorphic syndrome of hereditary neuralgic amyotrophy associated with a SEPT9 gene mutation--a family study.

We report a family in which two siblings presented with an apparent dysmorphic syndrome, including hypotelorism, blepharophimosis, slight ptosis, epicanthal folds, microstomia and dysmorphic ears. One sibling had a cleft palate. Initially, blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) was suspected; however, mutation of the FOXL2 gene was not detected. Moreover, the patients' father and paternal grandmother had experienced recurrent episodes of unilateral brachial neuritis and were diagnosed to have hereditary neuralgic amyotrophy (HNA). HNA is a rare, inherited form of brachial neuritis whose phenotypic spectrum may include hypotelorism, cleft palate and other minor dysmorphisms. HNA maps to chromosome 17q25 and is associated with mutations in the SEPT9 gene. After confirming a heterozygous SEPT9 mutation (R88W) in the father and his mother, it became apparent that the dysmorphic features in the children were part of HNA and that previous complaints of the daughter, erroneously diagnosed as pronatio dolorosa and then epiphysiolysis of the capitellum humeri, were in fact a first neuralgic pain attack. Both children were shown to have inherited the paternal SEPT9 mutation. Wider recognition of HNA as a syndromic disorder may facilitate its diagnosis in affected young persons who may not yet have manifested episodes of brachial neuritis.

An intragenic deletion of the factor IX gene in a family with hemophilia B.

A family of seven patients severely afflicted with hemophilia B has been studied for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (less than 10% of normal) factor IX antigen in urine and no detectable inhibitors in sera to factor IX protein. Based on the DNA hybridization analysis, these patients showed a partial intragenic deletion in their factor IX gene. The deletion included two exons (exons V and VI) coding for the amino acid sequence from number 85 to 195 of the factor IX protein. The deleted portion of the gene contained the entire factor IX activation peptide. The length of the deletion was estimated to be 10 +/- 0.3 kilobase pairs. This specific gene has been named FIXSeattle. In this family both the deletion and a Taq 1 restriction fragment length polymorphism can be used as a useful marker for accurate detection of female carriers of the deficient factor IX gene.

AHI1 mutations cause both retinal dystrophy and renal cystic disease in Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is an autosomal recessive disorder characterised by hypotonia, ataxia, mental retardation, altered respiratory pattern, abnormal eye movements, and a brain malformation known as the molar tooth sign (MTS) on cranial MRI. Four genetic loci have been mapped, with two genes identified (AHI1 and NPHP1). METHODS: We screened a cohort of 117 JS subjects for AHI1 mutations by a combination of haplotype analysis and sequencing of the gene, and for the homozygous NPHP1 deletion by sequencing and marker analysis. RESULTS: We identified a total of 15 novel AHI1 mutations in 13 families, including nonsense, missense, splice site, and insertion mutations, with some clustering in the WD40 domains. Eight families were consanguineous, but no single founder mutation was apparent. In addition to the MTS, retinal dystrophy was present in 11 of 12 informative families; however, no subjects exhibited variable features of JS such as polydactyly, encephalocele, colobomas, or liver fibrosis. In contrast to previous reports, we identified two families with affected siblings who developed renal disease consistent with nephronophthisis (NPH) in their 20s. In addition, two individuals with classic NPH were found to have homozygous NPHP1 deletions. CONCLUSIONS: Overall, 11% of subjects had AHI1 mutations, while approximately 2% had the NPHP1 deletion, representing a total of less than 15% in a large JS cohort. Some preliminary genotype-phenotype correlations are possible, notably the association of renal impairment, specifically NPH, in those with NPHP1 deletions. Subjects with AHI1 mutations may be at risk of developing both retinal dystrophy and progressive kidney disease.

Inherited neuropathies: from gene to disease.

Inherited disorders of peripheral nerves represent a common group of neurologic diseases. Charcot-Marie-Tooth neuropathy type 1 (CMT1) is a genetically heterogeneous group of chronic demyelinating polyneuropathies with loci mapping to chromosome 17 (CMT1A), chromosome 1 (CMT1B) and to another unknown autosome (CMT1C). CMT1A is most often associated with a tandem 1.5-megabase (Mb) duplication in chromosome 17p11.2-12, or in rare patients may result from a point mutation in the peripheral myelin protein-22 (PMP22) gene. CMT1B is associated with point mutations in the myelin protein zero (P0 or MPZ) gene. The molecular defect in CMT1C is unknown. X-linked Charcot-Marie-Tooth neuropathy (CMTX), which has clinical features similar to CMT1, is associated with mutations in the connexin32 gene. Charcot-Marie-Tooth neuropathy type 2 (CMT2) is an axonal neuropathy, also of undetermined cause. One form of CMT2 maps to chromosome 1p36 (CMT2A), another to chromosome 3p (CMT2B) and another to 7p (CMT2D). Dejerine-Sottas disease (DSD), also called hereditary motor and sensory neuropathy type III (HMSNIII), is a severe, infantile-onset demyelinating polyneuropathy syndrome that may be associated with point mutations in either the PMP22 gene or the P0 gene and shares considerable clinical and pathological features with CMT1. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder that results in a recurrent, episodic demyelinating neuropathy. HNPP is associated with a 1.5-Mb deletion in chromosome 17p11.2-12 and results from reduced expression of the PMP22 gene. CMT1A and HNPP are reciprocal duplication/deletion syndromes originating from unequal crossover during germ cell meiosis. Other rare forms of demyelinating peripheral neuropathies map to chromosome 8q, 10q and 11q. Hereditary neuralgic amyotrophy (familial brachial plexus neuropathy) is an autosomal dominant disorder causing painful, recurrent brachial plexopathies and maps to chromosome 17q25.

Comparison of single-strand conformation polymorphism and heteroduplex analysis for detection of mutations in Charcot-Marie-Tooth type 1 disease and related peripheral neuropathies.

To compare the sensitivity of the mutation detection techniques single-strand conformation polymorphism analysis (SSCP) and heteroduplex analysis (HA), we analyzed a cohort of 73 patients with a diagnosis of a demyelinating neuropathy, but without the CMT1A duplication, for mutations in the coding region of the myelin genes PMP22, MPZ and Cx32. In total, 21 samples showed 13 distinct altered migration patterns by one or both methods. Ten altered patterns were detected by both SSCP and HA, two were false negative by HA, and one was false negative by SSCP. Our results suggest that either technique can be useful for mutation detection, but a combination of factors appears to affect the sensitivity of both techniques.

Charcot-Marie-Tooth syndrome.

Charcot-Marie-Tooth syndrome (CMT) is a group of genetically determined symmetric distal polyneuropathies. The CMT loci are known to map to chromosome 1 (CMT1B), chromosome 17 (CMT1A), the X chromosome (CMTX), and two additional unknown autosomes (CMT1C and CMT2). The most prevalent form is CMT1A, an autosomal dominant demyelinative disorder caused either by a tandem duplication in band p11.2-12 of chromosome 17 (17p11.2-12) with trisomic expression of the peripheral myelin protein-22 (PMP-22) gene or, less frequently, by a missense mutation of PMP-22. Missense mutations in PMP-22 are also responsible for two forms of demyelinative polyneuropathy in mice, trembler and trembler. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent focal neuropathy. In all families thus far studied, patients with HNPP have been found to be monosomic for a segment of chromosome 17p11.2-12. The duplication in CMT1A and deletion in HNPP map to the same region in 17p11.2-12 and are both likely to be consequences of unequal crossing over during germ cell meiosis.

Genetic heterogeneity in Charcot-Marie-Tooth neuropathy type 2.

Charcot-Marie-Tooth neuropathy type 2 (CMT2) is a common inherited axonal neuropathy. The locus for one form of CMT2 (CMT2A) is assigned to the short arm of chromosome 1. There is genetic heterogeneity in CMT2 because additional pedigrees do not demonstrate linkage to chromosome 1 and are designated as CMT2B. Further clinical heterogeneity is suggested by CMT2 pedigrees with diaphragm and vocal cord weakness and are designated as CMT2C. To address the possible genetic distinction between CMT2A and CMT2C, we tested markers from the CMT2A locus for linkage in a large CMT2C pedigree. There was no evidence to support linkage of the CMT2C gene to the region of the CMT2A locus on chromosome 1. CMT2C is not an allelic variant of CMT2A. This analysis provides further evidence for genetic heterogeneity within inherited axonal neuropathies.

Genetic linkage relationships of Charcot-Marie-Tooth disease (HMSN-Ib) to chromosome 1 markers.

Hereditary motor and sensory neuropathy-Ib (HMSN-Ib) is a common autosomal dominant disorder linked to the Duffy blood group locus on human chromosome 1. The gene for antithrombin III (AT3) is also located on the long arm of chromosome 1. Using a DNA restriction fragment length polymorphism for AT3, we have investigated the genetic linkage relationship of all three markers (HMSN-Ib, Duffy, and AT3) in two affected families. Neither HMSN-Ib nor Duffy was tightly linked to AT3. The loci for both HMSN-Ib and Duffy must be close to the centromere on chromosome 1, but precise localization and gene order require study of additional markers and more families.

Analysis of the DNA duplication 17p11.2 in Charcot-Marie-Tooth neuropathy type 1 pedigrees: additional evidence for a third autosomal CMT1 locus.

We have restudied two clinically typical Charcot-Marie-Tooth neuropathy type 1 (CMT1; also known as hereditary motor and sensory neuropathy 1) pedigrees that were previously reported to be unlinked to the regions of proximal chromosome 1q and chromosome 17p by multipoint linkage analyses. In these two pedigrees, there is no evidence for linkage to additional DNA markers that flank and span the CMT1A locus on chromosome 17p11.2, and a duplication associated with CMT1A is not present in these pedigrees. These findings confirm that the CMT1 locus in these two pedigrees does not map to chromosome 17p11.2 or 1q, and provide further evidence for the existence of a third autosomal locus for CMT1.