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.

Genotype and clinical care correlations in craniosynostosis: findings from a cohort of 630 Australian and New Zealand patients.

Craniosynostosis is one of the most common craniofacial disorders encountered in clinical genetics practice, with an overall incidence of 1 in 2,500. Between 30% and 70% of syndromic craniosynostoses are caused by mutations in hotspots in the fibroblast growth factor receptor (FGFR) genes or in the TWIST1 gene with the difference in detection rates likely to be related to different study populations within craniofacial centers. Here we present results from molecular testing of an Australia and New Zealand cohort of 630 individuals with a diagnosis of craniosynostosis. Data were obtained by Sanger sequencing of FGFR1, FGFR2, and FGFR3 hotspot exons and the TWIST1 gene, as well as copy number detection of TWIST1. Of the 630 probands, there were 231 who had one of 80 distinct mutations (36%). Among the 80 mutations, 17 novel sequence variants were detected in three of the four genes screened. In addition to the proband cohort there were 96 individuals who underwent predictive or prenatal testing as part of family studies. Dysmorphic features consistent with the known FGFR1-3/TWIST1-associated syndromes were predictive for mutation detection. We also show a statistically significant association between splice site mutations in FGFR2 and a clinical diagnosis of Pfeiffer syndrome, more severe clinical phenotypes associated with FGFR2 exon 10 versus exon 8 mutations, and more frequent surgical procedures in the presence of a pathogenic mutation. Targeting gene hot spot areas for mutation analysis is a useful strategy to maximize the success of molecular diagnosis for individuals with craniosynostosis.

Co-occurrence of Joubert syndrome and Jeune asphyxiating thoracic dystrophy.

Ciliary disorders share typical features, such as polydactyly, renal and biliary cystic dysplasia, and retinitis pigmentosa, which often overlap across diagnostic entities. We report on two siblings of consanguineous parents and two unrelated children, both of unrelated parents, with co-occurrence of Joubert syndrome and Jeune asphyxiating thoracic dystrophy, an association that adds to the observation of common final patterns of malformations in ciliary disorders. Using homozygosity mapping in the siblings, we were able to exclude all known genes/loci for both syndromes except for INVS, AHI1, and three genes from the previously described Jeune locus at 15q13. No pathogenic variants were found in these genes by direct sequencing. In the third child reported, sequencing of RPGRIP1L, ARL13B, AHI1, TMEM67, OFD1, CC2D2A, and deletion analysis of NPHP1 showed no mutations. Although this study failed to identify a mutation in the patients tested, the co-occurrence of Joubert and Jeune syndromes is likely to represent a distinct entity caused by mutations in a yet to be discovered gene. The mechanisms by which certain organ systems are affected more than others in the spectrum of ciliary diseases remain largely unknown.

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.

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.

Genetic heterogeneity for autosomal recessive pyridoxine-dependent seizures.

Pyridoxine-dependent seizure (PDS) is a rare autosomal recessive intractable seizure disorder only controlled by a daily supplementation of pharmacological doses of pyridoxine (Vitamin B6). Although glutamate decarboxylase utilizes pyridoxal phosphate as a cofactor during conversion of the excitatory amino acid, glutamate, to the inhibitory neurotransmitter, gamma-amino butyric acid (GABA), several studies have failed to demonstrate a linkage to either of the glutamate-decarboxylase-encoding genes (GAD1 and GAD2) and PDS excluding involvement of this functional candidate. However, in 2000, a locus for PDS was mapped to a 5 cM interval at chromosome 5q31 in four consanguineous and one multisib pedigree (Z(max)=8.43 at theta=0 for marker D5S2017) [Cormier-Daire et al. in Am J Hum Genet 67(4):991-993 2000]. We undertook molecular genetic studies of six nonconsanguineous North American families, using up to ten microsatellite markers to perform haplotype segregation analysis of the 5q31 locus. Assignment to the chromosome 5q PDS locus was excluded in one of the six North American PDS pedigrees, as chromosome 5q31 haplotypes were incompatible with linkage to this locus. The remaining five PDS pedigrees showed haplotype segregation consistent with linkage to 5q31, generating a maximum combined lod score of 1.87 (theta=0) at marker D5S2011. In this study, we establish genetic heterogeneity for PDS, catalog 21 genes within the originally defined PDS interval, and identify additional recombinations that indicate a higher priority interval, containing just 11 genes.

Expression of SHOX in human fetal and childhood growth plate.

Abnormalities in the growth plate may lead to short stature and skeletal deformity including Leri Weil syndrome, which has been shown to result from deletions or mutations in the SHOX gene, a homeobox gene located at the pseudoautosomal region of the X and Y chromosome. We studied the expression of SHOX protein, by immunohistochemistry, in human fetal and childhood growth plates and mRNA by in situ hybridization in childhood normal and Leri Weil growth plate. SHOX protein was found in reserve, proliferative, and hypertrophic zones of fetal growth plate from 12 wk to term and childhood control and Leri Weil growth plates. The pattern of immunostaining in the proliferative zone of childhood growth plate was patchy, with more intense uniform immunostaining in the hypertrophic zone. In situ hybridization studies of childhood growth plate demonstrated SHOX mRNA expression throughout the growth plate. No difference in the pattern of SHOX protein or mRNA expression was seen between the control and Leri Weil growth plate. These findings suggest that SHOX plays a role in chondrocyte function in the growth plate.

Effect of 24 months of recombinant growth hormone on height and body proportions in SHOX haploinsufficiency.

Leri-Weill syndrome (LWS) is a skeletal dysplasia with mesomelic short stature, bilateral Madelung deformity (BMD) and SHOX (short stature homeobox-containing gene) haploinsufficiency. The effect of 24 months of recombinant human growth hormone (rhGH) therapy on the stature and BMD of two females with SHOX haploinsufficiency (demonstrated by fluorescence in situ hybridisation) and LWS was evaluated. Both patients demonstrated an increase in height standard deviation score (SDS) and height velocity SDS over the 24 months of therapy. Patient 1 demonstrated a relative increase in arm-span and upper segment measurements with rhGH while patient 2 demonstrated a relative increase in lower limb length. There was appropriate advancement of bone age, no adverse events and no significant deterioration in BMD. In this study, 24 months of rhGH was a safe and effective therapy for the disproportionate short stature of SHOX haploinsufficiency, with no clinical deterioration of BMD.

Familial growth and skeletal features associated with SHOX haploinsufficiency.

This study was designed to determine the intrafamilial effect of SHOX haploinsufficiency on stature, by comparing the growth and phenotype of 26 SHOX haploinsufficient individuals with 45 relatives and population standards. It confirmed that SHOX haploinsufficiency leads to growth restriction from birth to final height. Compared to unaffected siblings, the SHOX haploinsufficient cohort was 2.14 SDS (3.8 cm) shorter at birth and 2.1 SDS shorter through childhood. At final height females were 2.4 SDS (14.4 cm) shorter and males 0.8 SDS (5.3 cm) shorter than normal siblings. The family height analysis suggests that the effect of SHOX haploinsufficiency on growth may have been previously underestimated at birth and overestimated in males at final height. SHOX haploinsufficiency leads to short arms in 92%, bilateral Madelung deformity in 73% and short stature in 54%. Females were more severely affected than males. We conclude that SHOX is a major growth gene and that mutations are associated with a broad range of phenotype.

Multiple fetal anomalies associated with subtle subtelomeric chromosomal rearrangements.

We report two cases of multiple fetal anomalies detected by prenatal ultrasound and associated with subtle subtelomeric chromosomal rearrangements. The first case presented at 25 weeks of gestation with an enlarged cisterna magna and ventriculomegaly. Karyotyping of amniocytes showed a subtle terminal abnormality of chromosome 6q. Thereafter, screening of all unique chromosomal subtelomeric regions using a panel of telomere-specific, fluorescence in situ hybridization (FISH) probes revealed an unbalanced reciprocal translocation between 6q and 17p [46,XX.ish der(6)t(6;17)(q25.3;p13)(TelVysion6q-;TelVysion17p+)]. The second case presented at 25 weeks of gestation with tetralogy of Fallot and at 34 weeks of gestation had additional ultrasound findings of an arachnoid cyst and intrauterine growth restriction. Postnatal karyotyping of peripheral blood was performed and appeared normal. However, a cryptic deletion of the subtelomeric region of the long arm of chromosome 14 was identified when the infant's blood sample was used as a control for an oncology FISH probe. Thereafter, screening of all unique chromosomal subtelomeric regions using a panel of telomere-specific FISH probes revealed an unbalanced reciprocal translocation of chromosomes 14q and 20p [46,XY.ish der(14)t(14;20)(q32.3;p13)(IGH-, D14S308-,TelVysion20p+)mat]. These two cases add to a growing number of reports of cryptic subtelomeric chromosomal rearrangements associated with congenital anomalies. This is the first report of multiple, simultaneous FISH screening of the subtelomeric regions in amniotic fluid and has demonstrated the technical feasibility of this technique in the prenatal period.

Trisomy of the short stature homeobox-containing gene (SHOX), resulting from a duplication-deletion of the X chromosome.

The Turner syndrome (TS) is a complex disorder associated with almost invariant short stature and gonadal dysgenesis, as well as a variety of other major organ malformations. Recently, a homeobox-containing gene entitled short-stature homeobox-containing gene (SHOX), was isolated from a minimal short stature gene interval from the pseudoautosomal region of Xp (and Yp). Together with the demonstrable escape of SHOX from X-inactivation, this suggested SHOX to be a strong candidate gene for the short stature component of TS, and as SHOX haploinsufficiency appears to be the molecular basis of a mesomelic short statured skeletal dysplasia (Leri-Weill syndrome), this suggested that SHOX protein expression levels may confer a dosage effect on human stature. However, in this communication we report a normal statured female with gonadal dysgenesis, due to the inheritance of a recombinant duplication-deletion X-chromosome. The karyotype of the proband was 46,X,rec(X)dup(Xp)inv(X)(p11.22q21.2)mat and fluorescent in situ hybridization of her metaphases with a SHOX cosmid confirmed the proband to be trisomic for SHOX. This communication suggests the relationship between levels of SHOX expression and human stature to be more complex than envisaged previously. The presence of normal stature in our patient rather than tall stature is likely to represent the natural variation seen in patients with transcription factor disorders.

Histopathological analysis of Leri-Weill dyschondrosteosis: disordered growth plate.

Leri-Weill syndrome (LWS) is a dominant (pseudoautosomal) skeletal dysplasia with mesomelic short stature and bilateral Madelung deformity, due to dyschondrosteosis of the distal radius. It results from the loss of one copy of the Short Stature Homeobox Gene (SHOX) from the tip of the short arm of the X or Y chromosome. SHOX molecular testing enabled us to evaluate the histopathology of the radial physis in LWS patients with a documented SHOX abnormality. A widespread disorganisation of physeal anatomy was revealed with disruption of the normal parallel columnar arrangement of chondrocytes. Tandem stacking of maturing chondrocytes within columns was replaced by a side-by-side arrangement. The presence of hypertrophic osteoid with micro-enchondromata in the radial metaphysis suggests abnormal endochondral ossification. The Vickers' ligament was confirmed to blend with the triangular fibrocartilage complex (TFCC). This histopathological study demonstrates that the zone of dyschondrosteosis in LWS is characterised by marked disruption of normal physeal chondrocyte processes and that a generalised physeal abnormality is present.

Height discordance in monozygotic females is not attributable to discordant inactivation of X-linked stature determining genes.

We tested the hypothesis that X-linked genes determining stature which are subject to skewed or non-random X-inactivation can account for discordance in height in monozygotic female twins. Height discordant female monozygotic adult twins (20 pairs) were identified from the Australian Twin Registry, employing the selection criteria of proven monozygosity and a measured height discordance of at least 5 cm. Differential X-inactivation was examined in genomic DNA extracted from peripheral lymphocytes by estimating differential methylation of alleles at the polymorphic CAG triplet repeat of the Androgen receptor gene (XAR). There were 17/20 MZ pairs heterozygous at this locus and informative for analysis. Of these, 10/17 both had random X-inactivation, 5/17 showed identical X-inactivation patterns of non random inactivation and 2/17 (12%) showed discordant X-inactivation. There was no relationship between inactivation patterns and self-report chorionicity. We conclude that non-random X-inactivation does not appear to be a major contributor to intra-pair height discordance in female MZ twins.

Premature calvarial synostosis and epidermal hyperplasia (Beare-Stevenson syndrome-like anomalies) resulting from a P250R missense mutation in the gene encoding fibroblast growth factor receptor 3.

We report on a patient with a severe premature calvarial synostosis and epidermal hyperplasia. The phenotype was consistent with that of a mild presentation of Beare-Stevenson syndrome but molecular analysis of the IgIII-transmembrane linker region and the transmembrane domain of the gene encoding the FGFR2 receptor, revealed wild-type sequence only. Subsequently, molecular analysis of the FGFR3 receptor gene identified a heterozygous P250R missense mutation in both the proposita and her mildly affected father. This communication extends the clinical spectrum of the FGFR3 P250R mutation to encompass epidermal hyperplasia and documents the phenomenon of activated FGFR receptors stimulating common downstream developmental pathways, resulting in overlapping clinical outcomes.

The molecular basis of X-linked spondyloepiphyseal dysplasia tarda.

The X-linked form of spondyloepiphyseal dysplasia tarda (SEDL), a radiologically distinct skeletal dysplasia affecting the vertebrae and epiphyses, is caused by mutations in the SEDL gene. To characterize the molecular basis for SEDL, we have identified the spectrum of SEDL mutations in 30 of 36 unrelated cases of X-linked SEDL ascertained from different ethnic populations. Twenty-one different disease-associated mutations now have been identified throughout the SEDL gene. These include nonsense mutations in exons 4 and 5, missense mutations in exons 4 and 6, small (2-7 bp) and large (>1 kb) deletions, insertions, and putative splicing errors, with one splicing error due to a complex deletion/insertion mutation. Eight different frameshift mutations lead to a premature termination of translation and account for >43% (13/30) of SEDL cases, with half of these (7/13) being due to dinucleotide deletions. Altogether, deletions account for 57% (17/30) of all known SEDL mutations. Four recurrent mutations (IVS3+5G-->A, 157-158delAT, 191-192delTG, and 271-275delCAAGA) account for 43% (13/30) of confirmed SEDL cases. The results of haplotype analyses and the diverse ethnic origins of patients support recurrent mutations. Two patients with large deletions of SEDL exons were found, one with childhood onset of painful complications, the other relatively free of additional symptoms. However, we could not establish a clear genotype/phenotype correlation and therefore conclude that the complete unaltered SEDL-gene product is essential for normal bone growth. Molecular diagnosis can now be offered for presymptomatic testing of this disorder. Appropriate lifestyle decisions and, eventually, perhaps, specific SEDL therapies may ameliorate the prognosis of premature osteoarthritis and the need for hip arthroplasty.

Prenatal diagnosis of dominant dystrophic epidermolysis bullosa, by COL7A1 molecular analysis.

We report the first direct molecular prenatal diagnosis, undertaken for the autosomal dominant form of dystrophic epidermolysis bullosa (DDEB). The proband had a moderately severe form of DDEB, with episodic blistering of skin and mucosal involvement. Diagnostic histopathological examination, using electron microscopy to evaluate skin from a fresh blister, demonstrated a zone of cleavage beneath the epidermal-dermal junction, thereby assigning the EB as dystrophic. DNA analysis of COL7A1, the gene encoding type VII collagen, identified a heterozygous transversion (G to A) in the triple helix domain (G2043R). For any subsequent pregnancy, the affected mother and the unaffected father of the proband requested prenatal prediction, which was thereafter carried out in DNA extracted from a chorionic villus sample obtained at 11 weeks of gestation. Restriction enzyme analysis of COL7A1 exons 73 and 74 amplified by PCR, demonstrated the presence of the G2043R mutation, and the pregnancy was subsequently terminated. Molecular analysis of DNA extracted from fetal tissues confirmed the prenatal prediction.

Mild phenotype in two siblings with distal monosomy 12p13.31-->pter.

We report two sibs with trisomy for the region 2p25.1--> pter and monosomy for the region 12p13.31--> pter, due to adjacent-1 segregation of a maternal balanced reciprocal translocation, 46,XX,t(2;12)(p25.1;p13.31). These sibs presented with a mild phenotype, but nevertheless showed features of each of the contributing aneusomies. Monosomy 12p has previously been considered to have a variable and indistinct phenotype. Comparison of these patients with previous reports showed that many features, including microcephaly, facial dysmorphia, developmental and growth delay and dental and digital anomalies are frequently associated with monosomy for 12p. Many of these features are common to other aneusomies, thereby mitigating against a distinct 12p monosomy syndrome at this time. However, the combination of digital and dental anomalies may suggest the presence of this particular monosomy. The proband and his sister had some of the more non-specific features of 2p trisomy syndrome, and comparison with previous reports suggested that the characteristic 2p trisomy syndrome is more usually associated with larger or more proximal trisomies of 2p.

Clinical findings in a patient with FGFR1 P252R mutation and comparison with the literature.

We report on a patient with the skeletal findings of Jackson-Weiss syndrome, who manifests only mild craniofacial anomalies. Molecular analysis of her fibroblast growth factor receptor 1 gene (FGFR1) identified a heterozygous P252R missense mutation, previously only reported with FGFR1-Pfeiffer syndrome like manifestations. Mutations in the immunoglobulin-like, II-III (IgII-III) linker region of FGFR1 and FGFR3 molecules may present as a skeletal dysplasia affecting the appendicular skeleton including, brachydactyly, short broad middle phalanges, phalangeal epiphyseal coning and broad halluces. This communication is a further example of the phenomenon of an activated FGFR molecule resulting in overlapping manifestations in FGFR syndromes.

Identification and characterization of hydroxymethylbilane synthase mutations causing acute intermittent porphyria: evidence for an ancestral founder of the common G111R mutation.

Acute intermittent porphyria (AIP), the most common hepatic porphyria, results from the half-normal activity of hydroxymethylbilane synthase (HMB-synthase; EC 4.3.1.8), the third enzyme in the heme biosynthetic pathway. Because life-threatening acute neurologic attacks of this autosomal dominant disease are triggered by various ecogenic factors (e.g., certain drugs, hormones, alcohol, and starvation), efforts have been directed to identify and counsel presymptomatic heterozygotes in affected families to avoid the precipitating factors. Thus, to determine the nature of the mutations causing AIP in 26 unrelated enzyme-confirmed patients from Argentina, a long-range polymerase chain reaction method was developed to amplify the entire 10-kb gene in two fragments for efficient cycle sequencing and mutation detection. Eight new mutations were identified including two missense mutations (Q34P and G335S), four small deletions (728delCT, 815delAGGA, 948delA, and 985del12), a single base insertion (666insA), and a splice site mutation (IVS12(+1)). In addition, five previously reported mutations (G111R, R173W, Q204X, R201W, and 913insC) were detected. Notably, G111R was identified in 12 of the 26 (46%) presumably unrelated propositi; however, haplotype analysis with intragenic and flanking markers indicated an ancestral founder. Expression of the two new missense mutations (Q34P and G335S) in f1 E. coli resulted in 2.5% or less of the normal expressed enzyme, confirming their defective function. Thus, eight new and five previously reported HMB-synthase mutations, including a common lesion, were detected, permitting accurate identification and counseling of presymptomatic carriers in these 26 unrelated Argentinean AIP families with this dominant porphyria.