A founder nonsense variant in NUDT2 causes a recessive neurodevelopmental disorder in Saudi Arab children.

We identified the homozygous p.Arg12* variant in 5 patients with neurodevelopmental delay, but variation databases list many truncating heterozygous variants for this small 2-exon gene. As most of these affect the protein's C-terminus, loss-of-function mediated pathogenicity may be confined to bi-allelic truncating variants in exon 1 (nonsense-mediated decay!) or in the catalytically active Nudix box.

Expanding the clinical and genetic spectra of NKX6-2-related disorder.

Hypomyelinating leukodystrophies (HLDs) affect the white matter of the central nervous system and manifest as neurological disorders. They are genetically heterogeneous. Very recently, biallelic variants in NKX6-2 have been suggested to cause a novel form of autosomal recessive HLD. Using whole-exome or whole-genome sequencing, we identified the previously reported c.196delC and c.487C>G variants in NKX6-2 in 3 and 2 unrelated index cases, respectively; the novel c.608G>A variant was identified in a sixth patient. All variants were homozygous in affected family members only. Our patients share a primary diagnosis of psychomotor delay, and they show spastic quadriparesis, nystagmus and hypotonia. Seizures and dysmorphic features (observed in 2 families each) represent an addition to the phenotype, while developmental regression (observed in 3 families) appears to be a notable and previously underestimated clinical feature. Our findings extend the clinical and mutational spectra associated with this novel form of HLD. Comparative analysis of our 10 patients and the 15 reported previously did, however, not reveal clear evidence for a genotype-phenotype correlation.

Defective Connective Tissue Remodeling in Smad3 Mice Leads to Accelerated Aneurysmal Growth Through Disturbed Downstream TGF-ß Signaling.

Aneurysm-osteoarthritis syndrome characterized by unpredictable aortic aneurysm formation, is caused by SMAD3 mutations. SMAD3 is part of the SMAD2/3/4 transcription factor, essential for TGF-ß-activated transcription. Although TGF-ß-related gene mutations result in aneurysms, the underlying mechanism is unknown. Here, we examined aneurysm formation and progression in Smad3-/- animals. Smad3-/- animals developed aortic aneurysms rapidly, resulting in premature death. Aortic wall immunohistochemistry showed no increase in extracellular matrix and collagen accumulation, nor loss of vascular smooth muscle cells (VSMCs) but instead revealed medial elastin disruption and adventitial inflammation. Remarkably, matrix metalloproteases (MMPs) were not activated in VSMCs, but rather specifically in inflammatory areas. Although Smad3-/- aortas showed increased nuclear pSmad2 and pErk, indicating TGF-ß receptor activation, downstream TGF-ß-activated target genes were not upregulated. Increased pSmad2 and pErk staining in pre-aneurysmal Smad3-/- aortas implied that aortic damage and TGF-ß receptor-activated signaling precede aortic inflammation. Finally, impaired downstream TGF-ß activated transcription resulted in increased Smad3-/- VSMC proliferation. Smad3 deficiency leads to imbalanced activation of downstream genes, no activation of MMPs in VSMCs, and immune responses resulting in rapid aortic wall dilatation and rupture. Our findings uncover new possibilities for treatment of SMAD3 patients; instead of targeting TGF-ß signaling, immune suppression may be more beneficial.

Identification and analysis of a SMAD3 cis-acting eQTL operating in primary osteoarthritis and in the aneurysms and osteoarthritis syndrome.

OBJECTIVE: The TGF-ß pathway plays a central role in joint development with polymorphism in TGF-ß pathway genes implicated in osteoarthritis susceptibility. One association is to rs12901499, within intron 1 of SMAD3. Since rs12901499 is not in linkage disequilibrium with a non-synonymous polymorphism, it is likely the association is operating by influencing expression of SMAD3. DESIGN: Using tissues from the joints of primary osteoarthritis patients who had undergone joint replacement we measured the overall expression of SMAD3 by quantitative real-time PCR. We also measured allelic expression of SMAD3 using these tissues and vascular smooth muscle cells from patients with aneurysms and osteoarthritis syndrome, a rare condition featuring early-onset osteoarthritis. We tested the functional effect of SNPs in vitro using luciferase assays and assessed association with osteoarthritis using a large osteoarthritis case-control dataset. RESULTS: We observed that genotype at rs12901499 did not correlate with overall SMAD3 expression or allelic expression. However, genotype at a 3'UTR SNP, rs8031440, did correlate with SMAD3 expression in cartilage (P = 0.005) which was supported by allelic expression data showing that the G allele correlated with decreased SMAD3 expression in joint tissues and vascular smooth muscle cells. This G allele was underrepresented in osteoarthritis cases vs controls (P = 0.027, odds ratio = 0.921). rs8031440 is in perfect linkage disequilibrium with five other SMAD3 3'UTR SNPs and our luciferase analysis identified rs3743342 and rs12595334 as being functional. CONCLUSION: SMAD3 is subject to cis-acting regulatory polymorphism in the tissues of relevance to both primary osteoarthritis and the aneurysms-osteoarthritis syndrome.

A genome-wide linkage scan in a Dutch family identifies a premature ovarian failure susceptibility locus.

BACKGROUND: Premature ovarian failure (POF) is characterized by elevated gonadotrophins and amenorrhea before the age of 40 years and occurs approximately in 1% of women. POF etiology is highly heterogeneous with a wide spectrum of etiological pathogenic mechanisms including genetic causes. These mostly involve numerical, structural or monogenic defects on the X-chromosome. Mutations in a small number of autosomal genes (such as FOXL2 and NOBOX) have been identified as a cause of POF. However, in most cases, the disease underlying mechanisms are largely unknown. METHODS: We performed a genome-wide linkage analysis in a relatively large Dutch family with seven patients suffering from POF, showing a dominant pattern of inheritance. A genome-wide analysis, using 50K single nucleotide polymorphism arrays, was combined with conventional parametric linkage analysis. RESULTS: We identified three genomic regions on chromosomes 5, 14 and 18 yielding suggestive linkage (multipoint LOD score of 2.4 for each region). After inclusion of one elder unaffected family member, only the region on chromosome 5 remains as a putative POF locus. In addition, we investigated a second family (three living patients over three generations) for the regions on chromosome 5, 14 and 18. Haplotype analysis supported only the locus on chromosome 5q14.1-q15. CONCLUSION: We performed the first genome-wide linkage search in familial POF and identified a region on chromosome 5q14.1-q15, which may harbor a novel POF susceptibility gene.

STOX1 gene in pre-eclampsia and intrauterine growth restriction.

The STOX1 gene, identified as a candidate gene for pre-eclampsia in Dutch women, is placentally expressed and subject to imprinting with preferential transmission of the maternal allele. In our study, STOX1-Y153H frequencies were similar in 157 women with pre-eclampsia (65%) and in 157 controls (64%) from the general Dutch population. In an isolated Dutch population, a distortion could not be demonstrated in the transmission of STOX1-Y153H variation from heterozygous mothers to offspring in 50 and 56 families with pregnancies complicated by pre-eclampsia or intrauterine growth restriction, respectively. Our findings do not confirm previous suggestions that STOX1 plays a major role in Dutch women with pre-eclampsia.

A novel susceptibility locus for Hirschsprung's disease maps to 4q31.3-q32.3.

We report on a multigenerational family with isolated Hirschsprung's disease (HSCR). Five patients were affected by either short segment or long segment HSCR. The family consists of two main branches: one with four patients (three siblings and one maternal uncle) and one with one patient. Analysis of the RET gene, the major gene involved in HSCR susceptibility, revealed neither linkage nor mutations. A genome wide linkage analysis was performed, revealing suggestive linkage to a region on 4q31-q32 with a maximum parametric multipoint LOD score of 2.7. Furthermore, non-parametric linkage (NPL) analysis of the genome wide scan data revealed a NPL score of 2.54 (p = 0.003) for the same region on chromosome 4q (D4S413-D4S3351). The minimum linkage interval spans a region of 11.7 cM (12.2 Mb). No genes within this chromosomal interval have previously been implicated in HSCR. Considering the low penetrance of disease in this family, the 4q locus may be necessary but not sufficient to cause HSCR in the absence of modifying loci elsewhere in the genome. Our results suggest the existence of a new susceptibility locus for HSCR at 4q31.3-q32.3.

The G6055A (G2019S) mutation in LRRK2 is frequent in both early and late onset Parkinson's disease and originates from a common ancestor.

BACKGROUND: Mutations in the gene Leucine-Rich Repeat Kinase 2 (LRRK2) were recently identified as the cause of PARK8 linked autosomal dominant Parkinson's disease. OBJECTIVE: To study recurrent LRRK2 mutations in a large sample of patients from Italy, including early (<50 years) and late onset familial and sporadic Parkinson's disease. RESULTS: Among 629 probands, 13 (2.1%) were heterozygous carriers of the G2019S mutation. The mutation frequency was higher among familial (5.1%, 9/177) than among sporadic probands (0.9%, 4/452) (p<0.002), and highest among probands with one affected parent (8.7%, 6/69) (p<0.001). There was no difference in the frequency of the G2019S mutation in probands with early v late onset disease. Among 600 probands, one heterozygous R1441C but no R1441G or Y1699C mutations were detected. None of the four mutations was found in Italian controls. Haplotype analysis in families from five countries suggested that the G2019S mutation originated from a single ancient founder. The G2019S mutation was associated with the classical Parkinson's disease phenotype and a broad range of onset age (34 to 73 years). CONCLUSIONS: G2019S is the most common genetic determinant of Parkinson's disease identified so far. It is especially frequent among cases with familial Parkinson's disease of both early and late onset, but less common among sporadic cases. These findings have important implications for diagnosis and genetic counselling in Parkinson's disease.

A method for pooling alleles from different genotyping experiments.

Single tandem repeat (STR) polymorphisms are widely used in linkage and association studies. One of the drawbacks of using these markers is that genetic data coming from different experiments cannot be easily pooled together, because both allele length and binning distance may change. As large studies with multiple series of subjects sequentially included become more and more common, there is an increasing interest in pooling the genetic data obtained in different experiments. Correct reconstruction of allelic correspondences between genotyping experiments is particularly crucial for association-oriented studies, such as candidate gene studies and genome-wide association studies in isolated populations. Here, we suggest a maximum-likelihood framework to find the best correspondence between alleles typed in different genotyping experiments. We also address the issue of goodness-of-fit and robustness. We perform a study simulating results obtained in a genome scan using 787 STR markers. The simulations show that the suggested method yields good results with respect to the error rate, even if the sizes of the samples to be pooled are as low as 10 subjects (3% errors), though only 9% of alleles pass our tests. As sample sizes increase to 250 subjects the proportion of alleles pooled reaches 96% with an error rate of <0.1%.

Angiotensin converting enzyme gene polymorphism and cardiovascular morbidity and mortality: the Rotterdam Study.

BACKGROUND: Findings on the association between the insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene and cardiovascular morbidity and mortality have been inconsistent. Considering the possible interaction between this polymorphism and smoking, we evaluated the association between ACE I/D polymorphism and myocardial infarction (MI), mortality due to coronary heart disease (CHD), and cardiovascular disease (CVD). METHODS: The study was performed within the Rotterdam Study, a population based cohort study. The ACE I/D polymorphism was determined for 6714 participants and smoking status recorded at baseline. Fatal and non-fatal MIs and mortality events were regularly recorded. Cox proportional hazard analysis was performed separately for current smokers and non-smokers. We used age as the follow up time, presenting age specific survivals. RESULTS: During follow up, 248 MIs and 301 and 482 deaths, respectively, due to CHD and CVD occurred. There were no significant differences between the genotypes as regards MI incidence. Among smokers, there was an increased risk of CHD and CVD mortality in carriers of the DD genotype compared to the II genotype, which diminished at later ages (p<0.01 for gene-age interaction). Subgroup analysis in a younger and older group (based on the median age of 68.2 years) showed a significantly increased risk of CVD mortality in the younger group (hazard ratio = 5.19; 95% confidence interval: 1.15 to 23.42). CONCLUSIONS: This study showed that the ACE I/D polymorphism is not a strong risk factor for MI but its interaction with smoking might play a role in cardiovascular mortality especially at younger ages.

A study of gene--environment interaction on the gene for angiotensin converting enzyme: a combined functional and population based approach.

INTRODUCTION: Studies on the role of the insertion/deletion (I/D) polymorphism of the gene coding for angiotensin converting enzyme (ACE) in atherosclerosis have been inconsistent. In a meta-analysis, we recently showed that this relationship is stronger in high risk populations. In this paper, we used a combined functional and population based approach to investigate the gene-environment interaction of the ACE I/D polymorphism in relation to carotid artery wall thickness. METHODS: The study was part of the Rotterdam Study, a prospective population based cohort study. In 5321 subjects, IMT was measured in the carotid arteries by ultrasonography and ACE genotype was determined by size analysis of polymerase chain reaction products. RESULTS: In multiple regression analysis, I/D polymorphism and smoking were the main determinants for plasma ACE activity (r(2) = 0.28). There was a positive association between the D allele of the I/D polymorphism and carotid artery thickness among current smokers (p = 0.03). Subjects carrying only one of the risk factors (smoking or the D allele) did not show significant differences in IMT compared with the non-/former smokers group carrying two II alleles, while carriers of both risk factors had significant higher IMT. The association was not present in non-/former smokers. DISCUSSION: The results provide further evidence that genetic and environmental factors interact in the formation of the arterial lesions. This study shows that large population based studies can be extremely helpful in unravelling the genetic origin of complex diseases such as atherosclerosis.

A novel presenilin 1 mutation (L174 M) in a large Cuban family with early onset Alzheimer disease.

We studied a Cuban family with presenile dementia (autosomal dominant) consisting of 281 members within six generations, the proband descended from a Spanish founder. Mean age at onset was 59 years of age. Memory impairment was the main symptom in all patients, additionally, ischemic episodes were described in 4 (n = 18) patients. Neuropathological examination of brain material (1 patient) revealed neuronal loss, amyloid plaques, and neurofibrillary tangles. Thirty DNA samples were genotyped (regions on chromosome 1, 3, 10, 12, 14, 17, 19, 20, and 21). A maximum Lod score of 3.79 at theta = 0 was obtained for marker D14S43, located in a 9-cM interval in which all patients shared the same haplotype. Sequencing of the PSEN1 gene revealed a heterozygous base substitution, C520A (exon 6), which is predicted to cause an amino acid change from leucine to methionine in the TMIII of the presenilin 1 protein. The mutation was found to co-segregate with the disease phenotype and the associated disease haplotype. The C --> A change was not observed in 80 control chromosomes from the Cuban population. Leucine at position 174 is highly conserved among species and is identical in presenilin 1 and presenilin 2 proteins. We propose the L174 M mutation might lead to an abnormal N-terminal and probably C-terminal fragments and malfunction of the protein complex. In conclusion, we found a novel PSEN1 mutation in a large family with clinical and pathological diagnosis of early onset familial Alzheimer disease, which may be relevant for other Hispanic populations.