Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5pl5. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Autosomal dominant spondylocostal dysostosis is caused by mutation in TBX6

In humans, congenital spinal defects occur with an incidence of 0.5-1 per 1000 live births. One of the most severe syndromes with such defects is spondylocostal dysostosis (SCD). Over the past decade, the genetic basis of several forms of autosomal recessive SCD cases has been solved with the identification of four causative genes (DLL3, MESP2, LFNG and HES7). Autosomal dominant forms of SCD have also been reported, but to date no genetic etiology has been described for these. Here, we have used exome capture and next-generation sequencing to identify a stoploss mutation in TBX6 that segregates with disease in two generations of one family. We show that this mutation has a deleterious effect on the transcriptional activation activity of the TBX6 protein, likely due to haploinsufficiency. In mouse, Tbx6 is essential for the patterning of the vertebral precursor tissues, somites; thus, mutation of TBX6 is likely to be causative of SCD in this family. This is the first identification of the genetic cause of an autosomal dominant form of SCD, and also demonstrates the potential of exome sequencing to identify genetic causes of dominant diseases even in small families with few affected individuals.

The IFITM5 mutation c.-14C > T results in an elongated transcript expressed in human bone; And causes varying phenotypic severity of osteogenesis imperfecta type V

Background The genetic mutation resulting in osteogenesis imperfecta (OI) type V was recently characterised as a single point mutation (c.-14C > T) in the 5' untranslated region (UTR) of IFITM5, a gene encoding a transmembrane protein with expression restricted to skeletal tissue. This mutation creates an alternative start codon and has been shown in a eukaryotic cell line to result in a longer variant of IFITM5, but its expression has not previously been demonstrated in bone from a patient with OI type V. Methods Sanger sequencing of the IFITM5 5' UTR was performed in our cohort of subjects with a clinical diagnosis of OI type V. Clinical data was collated from referring clinicians. RNA was extracted from a bone sample from one patient and Sanger sequenced to determine expression of wild-type and mutant IFITM5. Results: All nine subjects with OI type V were heterozygous for the c.-14C > T IFITM5 mutation. Clinically, there was heterogeneity in phenotype, particularly in the manifestation of bone fragility amongst subjects. Both wild-type and mutant IFITM5 mRNA transcripts were present in bone. Conclusions The c.-14C > T IFITM5 mutation does not result in an RNA-null allele but is expressed in bone. Individuals with identical mutations in IFITM5 have highly variable phenotypic expression, even within the same family.

A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

Next-generation sequencing: A frameshift in skeletal dysplasia gene discovery

In the last decade, huge breakthroughs in genetics - driven by new technology and different statistical approaches - have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis. © International Osteoporosis Foundation and National Osteoporosis Foundation 2013.

Precipitazioni sull'endotelio [Pigment Dispersion Syndrome]

Arguably, the most common patient seen in contact lens practice in our communities is the young adult white myope. The incidence of eye disease in this group of patients is very low, particularly if the genetically determined problems are excluded. However, there is one condition that should always be anticipated and searched for, especially in males. In fact, it affects 2-4% of these individuals and is known as pigment dispersion syndrome. This is important because 25-50% of these patients will get secondary or pigmentary glaucoma because of the pigment dispersion. It can be inherited as an autosomal dominant trait and has been mapped to chromosome 7. It is usually bilateral and is rarely encountered in patients of darker skin such as Asians and African-Americans.

Detection of a novel mutation in the CACNA1A gene

Familial hemiplegic migraine (FHM) is a rare autosomal dominant subtype of migraine with aura. It is divided into three subtypes FHM1, FHM2 and FHM3, which are caused by mutations in the CACNA1A, ATP1A2 and SCN1A genes respectively. As part of a regular diagnostic service, we investigated 168 patients with FHM symptoms. Samples were tested for mutations contained within the CACNA1A gene. Some tested samples (4.43%) showed an FHM1 mutation, with five of the mutations found in exon 5, one mutation in exon 16 and one in exon 17. Four polymorphisms were also detected, one of which occurred in a large percentage of samples (14.88%). The exon 16 2094G>A polymorphism, however, has been found to occur in healthy Caucasian control populations up to a frequency of 16% and is not considered to be significantly associated with FHM. A finding of significance, found in a single patient, was the detection of a novel mutation in exon 5 that results in a P225H change. The affected individual was an 8-year-old female. The exact phenotypic effect of this mutation is unknown, and further studies are needed to understand the pathophysiology of this mutation in FHM1. New information will allow for diagnostic procedures to be constantly updated, thus improving accuracy of diagnosis. It is possible that new information will also aid the development of new therapeutic agents for the treatment of FHM.

Functional analysis of missense variants in the TRESK (KCNK18) K+ channel

A loss of function mutation in the TRESK K2P potassium channel (KCNK18), has recently been linked with typical familial migraine with aura. We now report the functional characterisation of additional TRESK channel missense variants identified in unrelated patients. Several variants either had no apparent functional effect, or they caused a reduction in channel activity. However, the C110R variant was found to cause a complete loss of TRESK function, yet is present in both sporadic migraine and control cohorts, and no variation in KCNK18 copy number was found. Thus despite the previously identified association between loss of TRESK channel activity and migraine in a large multigenerational pedigree, this finding indicates that a single non-functional TRESK variant is not alone sufficient to cause typical migraine and highlights the genetic complexity of this disorder. Migraine is a common, disabling neurological disorder with a genetic, environmental and in some cases hormonal component. It is characterized by attacks of severe, usually unilateral and throbbing headache, can be accompanied by nausea, vomiting and photophobia and is clinically divided into two main subtypes, migraine with aura (MA) when a migraine is accompanied by transient and reversible focal neurological symptoms and migraine without aura (MO)1. The multifactorial and clinical heterogeneity of the disorder have considerably hindered the identification of common migraine susceptibility genes and most of our current understanding comes from the studies of familial hemiplegic migraine (FHM), a rare monogenic autosomal dominant form of MA2. So far, the three susceptibility genes that have been convincingly identified in FHM families all encode ion channels or transporters: CACNA1A encoding the α1 subunit of the Cav2.1 calcium channel3, SCN1A encoding the Nav1.1 sodium channel4 and ATP1A2 encoding the α2 subunit of the Na+/K+ pump5. It is believed that mutations in these genes may lead to increased efflux of glutamate and potassium in the synapse and thereby cause migraine by rendering the brain more susceptible to cortical spreading depression (CSD)6 which is thought to play a role in initiating a migraine attack7,8. However, these genes have not to date been implicated in common forms of migraine9. Nevertheless, current opinion suggests that typical migraine, like FHM, is also disorder of neuronal excitability, ion homeostasis and neurotransmitter release10,11,12. Mutations in the SLC4A4 gene encoding the sodium-bicarbonate cotransporter NBCe1, have recently been implicated in several different forms of migraine13, and a variety of genes involved in glutamate homeostasis (PGCP, MTDH14 and LRP115) and a cation channel (TRPM8)15 have also recently been implicated in migraine via genome-wide association studies. Ion channels are therefore highly likely to play an important role in the pathogenesis of typical migraine. TRESK (KCNK18), is a member of the two-pore domain (K2P) family of potassium channels involved in the control of cellular electrical excitability16. Regulation of TRESK activity by the calcium-dependent phosphatase calcineurin17, as well as its expression in dorsal root ganglia (DRG)18 and trigeminal ganglia (TG)19,20 has led to a proposed role for this channel in a variety of pain pathways. In a recent study, a frameshift mutation (F139Wfsx24) in TRESK was identified in a large multigenerational pedigree where it co-segregated perfectly with typical MA and a significant genome-wide linkage LOD score of 3.0. Furthermore, functional analysis revealed that this mutation caused a complete loss of TRESK function and that the truncated subunit was also capable of down regulating wild-type channel function. This therefore highlighted KCNK18 as potentially important candidate gene and suggested that TRESK dysfunction might play a possible role in the pathogenesis of familial migraine with visual aura20. Additional screening for KCNK18 mutations in unrelated sporadic migraine and control cohorts also identified a number of other missense variants; R10G, A34V, C110R, S231P and A233V20. The A233V variant was found only in the control cohort, whilst A34V was identified in a single Australian migraine proband for which family samples were not available, but it was not detected in controls. By contrast, the R10G, C110R, and S231P variants were found in both migraineurs and controls in both cohorts. In this study, we have investigated the functional effect of these variants to further probe the potential association of TRESK dysfunction with typical migraine.

Two novel mutations and a previously unreported intronic polymorphism in the NOTCH3 gene

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease of small vessel caused by mutations in the NOTCH3 gene (NCBI Gene ID: 4854) located on chromosome 19p13.1. NOTCH3 consists of 33 exons which encode a protein of 2321 amino acids. Exons 3 and 4 were found to be mutation hotspots, containing more than 65% of all CADASIL mutations. We performed direct sequencing on an ABI 3130 Genetic Analyser to screen for mutations and polymorphisms on 300 patients who were clinically suspected to have CADASIL. First, exons 3 and 4 were screened in NOTCH3 and if there were no variations found, then extended CADASIL testing (exons 2, 11, 18 and 19) was offered to patients. Here we report two novel non-synonymous mutations identified in the NOTCH3 gene. The first mutation, located in exon 4 was found in a 49-year-old female and causes an alanine to valine amino acid change at position 202 (605C > T). The second mutation, located in exon 11, was found in a 66-year-old female and causes a cysteine to arginine amino acid change at position 579 (1735T > C). We also report a 46-year-old male with a known polymorphism Thr101Thr (rs3815188) and an unreported polymorphism NM_000435.2:c.679+60G>A observed in intron 4 of the NOTCH3 gene. Although Ala202Ala (rs1043994) is a common polymorphism in the NOTCH3 gene, our reported novel mutation (Ala202Val) causes an amino acid change at the same locus. Our other reported mutation (Cys579Arg) correlates well with other known mutations in NOTCH3, as the majority of the CADASIL-associated mutations in NOTCH3 generally occur in the EGF-like (epidermal growth factor-like) repeat domain, causing a change in the number of cysteine residues. The intronic polymorphism NM_000435.2:c.679+60G>A lies close to the intron–exon boundary and may affect the splicing mechanism in the NOTCH3 gene.

Association of a Notch 3 gene polymorphism with migraine susceptibility

Introduction Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) shares common symptoms with migraine. Most CADASIL causative mutations occur in exons 3 and 4 of the Notch 3 gene. This study investigated the role of C381T (rs 3815188) and G684A (rs 1043994) single nucleotide polymorphisms (SNP) in exons 3 and 4, respectively, of the Notch 3 gene in migraine. Results The first part of the study, in a population of 275 migraineurs and 275 control individuals, found a significant association between the C381T variant and migraine, specifically in migraine without aura (MO) sufferers. The G684A variant was also found to be significantly associated with migraine, specifically in migraine with aura (MA) sufferers. A follow-up study in 300 migraineurs and 300 control individuals did not show replicated association of the C381T variant with migraineurs. However, the G684A variant was again shown to be significantly associated with migraine, specifically with MA. Conclusion Further investigation of the G684A variant and the Notch 3 gene is warranted to understand their role in migraine.

Chromosome I linkage studies in Charcot-Marie-Tooth neuropathy type I

Charcot-Marie-Tooth neuropathy type 1 (CMT1) is an autosomal dominant disorder of peripheral nerve. The gene for CMT1 was originally localized to chromosome 1 by linkage to the Duffy blood group, but it has since been shown that not all CMT1 pedigrees show this linkage. We report here the results of linkage studies using five chromosome 1 markers - Duffy (Fy), antithrombin III (AT3), renin (REN), β-nerve growth factor (NGFB), and salivary amylase (AMY1) - in 16 CMT1 pedigrees. The total lod scores exclude close linkage of CMT1 to any of these markers. However, individual families show probable linkage of CMT1 to Duffy, AT3, and/or AMY1. No linkage was indicated with REN or NGFB. These results indicate that possible location of a CMT1 gene between the AMY1 and AT3 loci at p21 and q23, respectively, on chromosome 1 and support the theory that there is at least one other CMT1 gene.

Heterogeneity evidence and linkage studies on Charcot-Marie-Tooth disease

Charcot-Marie-Tooth neuropathy type 1 (CMT1) is an autosomal dominant disorder originally localized to chromosome 1 by linkage to the Duffy blood group. Studies have since shown that the disorder may be heterogeneous, as not all families show this linkage. We tested genetic heterogeneity by the HOMOG computer program in 15 CMT1 pedigrees informative for Duffy. We detected no evidence for heterogeneity in this sample, but when we combined results with previously published lod scores, heterogeneity was statistically significant. Twelve of the 15 families studied did not show linkage to Duffy. We found six of these families to be informative for a chromosome 19 marker, apolipoprotein CII(ApoC2). Despite a previous report showing probable linkage of a non-Duffy-linked CMT1 pedigree to two chromosome 19 markers, we did not detect significant linkage of ApoC2 to CMT1 in these families.

Association and linkage analyses of restriction fragment length polymorphisms for the human renin and antithrombin III genes in essential hypertension

Essential hypertension is a highly hereditable disorder in which genetic influences predominate over environmental factors. The molecular genetic profiles which predispose to essential hypertension are not known. In rats with genetic hypertension, there is some recent evidence pointing to linkage of renin gene alleles with blood pressure. The genes for renin and antithrombin III belong to a conserved synteny group which, in humans, spans the q21.3-32.3 region of chromosome I and, in rats, is linkage group X on chromosome 13. The present study examined the association of particular human renin gene (REN) and antithrombin III gene (AT3) polymorphisms with essential hypertension by comparing the frequency of specific alleles for each of these genes in 50 hypertensive offspring of hypertensive parents and 91 normotensive offspring of normotensive parents. In addition, linkage relationships were examined in hypertensive pedigrees with multiple affected individuals. Alleles of a REN HindIII restriction fragment length polymorphism (RFLP) were detected using a genomic clone, λHR5, to probe Southern blots of HindIII-cut leucocyte DNA, and those for an AT3 Pstl RFLP were detected by phATIII 113 complementary DNA probe. The frequencies of each REN allele in the hypertensive group were 0.76 and 0.24 compared with 0.74 and 0.26 in the normotensive group. For AT3, hypertensive allele frequencies were 0.49 and 0.51 compared with normotensive values of 0.54 and 0.46. These differences were not significant by χ2 analysis (P > 0.2). Linkage analysis of a family (data from 16 family members, 10 of whom were hypertensive), informative for both markers, without an age-of-onset correction, and assuming dominant inheritance of hypertension, complete penetrance and a disease frequency of 20%, did not indicate linkage of REN with hypertension, but gave a positive, although not significant, logarithm of the odds for linkage score of 0.784 at a recombination fraction of 0 for AT3 linkage to hypertension. In conclusion, the present study could find no evidence for an association of a REN HindIII RFLP with essential hypertension or for a linkage of the locus defined by this RFLP in a family segregating for hypertension. In the case of an AT3 Pstl RFLP, although association analysis was negative, linkage analysis suggested possible involvement (odds of 6:1 in favour) of a gene located near the 1q23 locus with hypertension in one informative family.

Visual function and CFH/ARMS2 risk genotypes in macular dystrophy due to maternally inherited diabetes and deafness

Maternally inherited diabetes and deafness (MIDD) is an autosomal dominant inherited syndrome caused by the mitochondrial DNA (mtDNA) nucleotide mutation A3243G. It affects various organs including the eye with external ophthalmoparesis, ptosis, and bilateral macular pattern dystrophy.1, 2 The prevalence of retinal involvement in MIDD is high, with 50% to 85% of patients exhibiting some macular changes.1 Those changes, however, can vary between patients and within families dramatically based on the percentage of retinal mtDNA mutations, making it difficult to give predictions on an individual’s visual prognosis...