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BACKGROUND: Sacral agenesis (SA) consists of partial or complete absence of the caudal end of the spine and often presents with additional birth defects. Several studies have examined gene variants for syndromic forms of SA, but only one has examined exomes of children with non-syndromic SA. METHODS: Using buccal cell specimens from families of children with non-syndromic SA, exomes of 28 child-parent trios (eight with and 20 without a maternal diagnosis of pregestational diabetes) and two child-father duos (neither with diagnosis of maternal pregestational diabetes) were exome sequenced. RESULTS: Three children had heterozygous missense variants in ID1 (Inhibitor of DNA Binding 1), with CADD scores >20 (top 1% of deleterious variants in the genome); two children inherited the variant from their fathers and one from the child’s mother. Rare missense variants were also detected in PDZD2 (PDZ Domain Containing 2; N=1) and SPTBN5 (Spectrin Beta, Non-erythrocytic 5; N=2), two genes previously suggested to be associated with SA etiology. Examination of variants with autosomal recessive and X-linked recessive inheritance identified five and two missense variants, respectively. Compound heterozygous variants were identified in several genes. In addition, 12 de novo variants were identified, all in different genes in different children. CONCLUSIONS: To our knowledge, this is the first study reporting a possible association between ID1 and non-syndromic SA. Although maternal pregestational diabetes has been strongly associated with SA, the missense variants in ID1 identified in two of three children were paternally inherited. These findings add to the knowledge of gene variants associated with non-syndromic SA and provide data for future studies.

The biological and clinical significance of the p.E88del variant in the transcobalamin receptor, CD320, is unknown. This allele is annotated in ClinVar as likely benign, pathogenic, and of uncertain significance. To determine functional consequence and clinical relevance of this allele, we employed cell culture and genetic association studies. Fibroblasts from sixteen CD320 p.E88del homozygotes exhibited reduced binding and uptake of cobalamin. Complete ascertainment of newborns with transiently elevated C3 (propionylcarnitine) in New York State demonstrated that homozygosity for CD320 p.E88del was over-represented (7/348, p

Bladder exstrophy (BE) is a rare, lower ventral midline defect with the bladder and part of the urethra exposed. The etiology of BE is unknown but thought to be influenced by genetic variation with more recent studies suggesting a role for rare variants. As such, we conducted paired-end exome sequencing in 26 child/mother/father trios. Three children had rare (allele frequency ≤ 0.0001 in several public databases) inherited variants in TSPAN4, one with a loss-of-function variant and two with missense variants. Two children had loss-of-function variants in TUBE1. Four children had rare missense or nonsense variants (one per child) in WNT3, CRKL, MYH9, or LZTR1, genes previously associated with BE. We detected 17 de novo missense variants in 13 children and three de novo loss-of-function variants (AKR1C2, PRRX1, PPM1D) in three children (one per child). We also detected rare compound heterozygous loss-of-function variants in PLCH2 and CLEC4M and rare inherited missense or loss-of-function variants in additional genes applying autosomal recessive (three genes) and X-linked recessive inheritance models (13 genes). Variants in two genes identified may implicate disruption in cell migration (TUBE1) and adhesion (TSPAN4) processes, mechanisms proposed for BE, and provide additional evidence for rare variants in the development of this defect.

Background Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. Methods We genotyped 32 HRHS cases identified from all New York State live births (1998–2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20 Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3, and Childrens Hospital of Philadelphia database. Results We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16-2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1−/− mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5 Mb deletion associated with Williams-Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24 Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. Conclusion To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. Birth Defects Research 109:16–26, 2017. © 2016 Wiley Periodicals, Inc.

Klippel-Trenaunay syndrome (KTS) is a rare congenital vascular disorder that is thought to occur sporadically; however, reports of familial occurrence suggest a genetic component. We examined KTS cases to identify novel, potentially causal copy number variants (CNVs). We identified 17 KTS cases from all live-births occurring in New York (1998-2010). Extracted DNA was genotyped using Illumina microarrays and CNVs were called using PennCNV software. CNVs selected for follow-up had ≥10 single nucleotide polymorphisms (SNPs) and minimal overlap with in-house controls or controls from the Database of Genomic Variants. We identified 15 candidate CNVs in seven cases; among them a deletion in two cases within transcripts of HDAC9, a histone deacetylase essential for angiogenic sprouting of endothelial cells. One of them also had a duplication upstream of SALL3, a transcription factor essential for embryonic development that inhibits DNMT3A, a DNA methyltransferase responsible for embryonic de novo DNA methylation. Another case had a duplication spanning ING5, a histone acetylation regulator active during embryogenesis. We identified rare genetic variants related to chromatin modification which may have a key role in regulating vascular development during embryogenesis. Further investigation of their implications in the pathogenesis of KTS is warranted. © 2016 Wiley Periodicals, Inc.

The cause of posterior urethral valves (PUV) is unknown, but genetic factors are suspected given their familial occurrence. We examined cases of isolated PUV to identify novel copy number variants (CNVs). We identified 56 cases of isolated PUV from all live-births in New York State (1998-2005). Samples were genotyped using Illumina HumanOmni2.5 microarrays. Autosomal and sex-linked CNVs were identified using PennCNV and cnvPartition software. CNVs were prioritized for follow-up if they were absent from in-house controls, contained ≥ 10 consecutive probes, were ≥ 20 Kb in size, had ≤ 20% overlap with variants detected in other birth defect phenotypes screened in our lab, and were rare in population reference controls. We identified 47 rare candidate PUV-associated CNVs in 32 cases; one case had a 3.9 Mb deletion encompassing BMP7. Mutations in BMP7 have been associated with severe anomalies in the mouse urethra. Other interesting CNVs, each detected in a single PUV case included: a deletion of PIK3R3 and TSPAN1, duplication/triplication in FGF12, duplication of FAT1--a gene essential for normal growth and development, a large deletion (>2 Mb) on chromosome 17q that involves TBX2 and TBX4, and large duplications (>1 Mb) on chromosomes 3q and 6q. Our finding of previously unreported novel CNVs in PUV suggests that genetic factors may play a larger role than previously understood. Our data show a potential role of CNVs in up to 57% of cases examined. Investigation of genes in these CNVs may provide further insights into genetic variants that contribute to PUV.

Summary Newborn screening for Cystic Fibrosis (CF) began in New York in October, 2002 using immunoreactive trypsinogen (IRT)/DNA methodology. Infants with at least one CFTR mutation or very high IRT and no mutations (VHIRT) are referred for sweat testing. In a preliminary analysis, we noted a very low positive predictive value (PPV) and preponderance of Hispanic infants in the group of infants with CF referred for VHIRT, which led to a decision to revise, but not eliminate, the VHIRT category. Automatic referral for specimens with VHIRT collected on the day of birth was eliminated, and the VHIRT threshold was raised from 0.2% to 0.1%. In this report, we describe outcomes from VHIRT referrals among 2.4 million infants screened between March 2003 and February 2013. Following the algorithm change, referrals decreased by 37.8% overall (annual mean 1,485 vs. 923), and the VHIRT PPV improved (0.6–1.0%). The number of infants diagnosed has remained consistent at 1 in 4,400 births. The proportion of Black/Hispanic/Asian/Other infants with confirmed CF, CFTR-related metabolic syndrome (CRMS), or possible CF/CRMS was 21.3% in infants with 1–2 mutations, but 75.8% in the VHIRT group. In conclusion, although the PPV among VHIRT referrals remains low, had this category never been implemented, 24 infants with confirmed CF, and 9 infants with CRMS or possible CF/CRMS, most of whom were Hispanic, would have been missed over the 10 years. Information from this study may be helpful in assessing the need for the VHIRT category and algorithm changes in other screening programs. Pediatr Pulmonol. 2015; 50:771–780. © 2015 Wiley Periodicals, Inc.

Anorectal atresia is a serious birth defect of largely unknown etiology but candidate genes have been identified in animal studies and human syndromes. Because alterations in the activity of these genes might lead to anorectal atresia, we selected 71 common variants predicted to be in transcription factor binding sites, CpG windows, splice sites, and miRNA target sites of 25 candidate genes, and tested for their association with anorectal atresia. The study population comprised 150 anorectal atresia cases and 623 control infants without major malformations. Variants predicted to affect transcription factor binding, splicing, and DNA methylation in WNT3A, PCSK5, TCF4, MKKS, GLI2, HOXD12, and BMP4 were associated with anorectal atresia based on a nominal P value < 0.05. The GLI2 and BMP4 variants are reported to be moderately associated with gene expression changes (Spearman's rank correlation coefficients between -0.260 and 0.226). We did not find evidence for interaction between maternal pre-pregnancy obesity and variants in MKKS, a gene previously associated with obesity, on the risk of anorectal atresia. Our results for MKKS support previously suggested associations with anorectal malformations. Our findings suggest that more research is needed to determine whether altered GLI2 and BMP4 expression is important in anorectal atresia in humans.

We conducted a population-based case-control study of single nucleotide polymorphisms (SNPs) in selected genes to find common variants that play a role in the etiology of limb deficiencies (LDs). Included in the study were 389 infants with LDs of unknown cause and 980 unaffected controls selected from all births in New York State (NYS) for the years 1998-2005. We used cases identified from the NYS Department of Health (DOH) Congenital Malformations Registry. Genotypes were obtained for 132 SNPs in genes involved in limb development (SHH, WNT7A, FGF4, FGF8, FGF10, TBX3, TBX5, SALL4, GREM1, GDF5, CTNNB1, EN1, CYP26A1, CYP26B1), angiogenesis (VEGFA, HIF1A, NOS3), and coagulation (F2, F5, MTHFR). Genotype call rates were97% and SNPs were tested for departure from Hardy-Weinberg expectations by race/ethnic subgroups. For each SNP, odds ratios (OR)s and confidence intervals (CI)s were estimated and corrected for multiple comparisons for all LDs combined and for LD subtypes. Among non-Hispanic white infants, associations between FGF10 SNPs rs10805683 and rs13170645 and all LDs combined were statistically significant following correction for multiple testing (OR = 1.99; 95% CI = 1.43-2.77; uncorrected P = 0.000043 for rs10805683 heterozygous genotype, and OR = 2.37; 95% CI = 1.48-3.78; uncorrected P = 0.00032 for rs13170645 homozygous minor genotype). We also observed suggestive evidence for associations with SNPs in other genes including CYP26B1 and WNT7A. Animal studies have shown that FGF10 induces formation of the apical ectodermal ridge and is necessary for limb development. Our data suggest that common variants in FGF10 increase the risk for a wide range of non-syndromic limb deficiencies.

Point mutations and copy number variations in SNCA, the gene encoding alpha-synuclein, cause familial Parkinson's disease (PD). A dinucleotide polymorphism (REP1) in the SNCA promoter may be a risk factor for common forms of PD. We studied 1,802 PD patients and 2,129 controls from the NeuroGenetics Research Consortium, using uniform, standardized protocols for diagnosis, subject recruitment, data collection, genotyping, and data analysis. Three common REP1 alleles (257, 259, and 261 bp, with control frequencies of 0.28, 0.65, and 0.06) and several rare alleles (combined frequency

UCHL1 has been proposed as a candidate gene for Parkinson’s disease (PD). A meta-analysis of white and Asian subjects reported an inverse association between the non-synonymous UCHL1 S18Y polymorphism and PD risk. However, this finding was not replicated in a large case–control study and updated meta-analysis restricted to white subjects. We performed a case–control study of 1757 PD patients recruited from movement disorder clinics and 2016 unrelated controls from four regions of the United States. All subjects self-reported as white. We did not observe evidence for an association between S18Y genotypes and PD (overall P-value for association: P = 0.42). After adjustment for age, sex, and recruitment region, the odds ratio for Y/S versus S/S was 0.91 (95% CI: 0.78–1.06) and for Y/Y versus S/S was 0.87 (95% CI: 0.58–1.29). We also did not observe a significant association for recessive or dominant models of inheritance, or after stratification by age at onset, age at blood draw, sex, family history of PD, or recruitment region. Our results suggest that UCHL1 S18Y is not a major susceptibility factor for PD in white populations although we cannot exclude the possibility that the S18Y variant exerts weak effects on risk, particularly in early-onset disease.

The microtubule-associated protein tau, encoded by the MAPT gene, is primarily expressed in neurons and plays a key role in the organization and integrity of the cytoskeleton.1,2 Filamentous neuronal tau inclusions define a set of neurodegenerative diseases collectively known as the tauopathies, which include Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and frontotemporal dementia with parkinsonism linked to chromosome 17.1,2 MAPT sequence variation was first linked to the etiopathogenesis of tauopathies by the discovery of mutations resulting in frontotemporal dementia with parkinsonism linked to chromosome 17, and subsequently an extended common haplotype (H1) across the gene was shown to associate with disease risk in PSP and CBD.3–7 Though Parkinson’s disease (PD) shares some clinical features with the tauopathies, it has been assigned to a distinct subset of neurodegenerative diseases (the α-synucleinopathies) by histopathology because it is characterized by intraneuronal accumulation of α-synuclein, rather than tau.8 Thus, initial reports that the MAPT H1 haplotype was also associated with PD were intriguing but met with some skepticism. Since then the association between MAPT variants and PD has remained tenuous with a number of small case–control studies reporting either a marginal or no significant overrepresentation of the H1/H1 diplotype among cases (Table 1).9–23However, a recent meta-analysis of 10 such studies in white subjects supported the hypothesis that the H1 haplotype might confer susceptibility for PD (pooled odds ratio [OR] for H1/H1 vs H1/H2 and H2/H2, 1.49; confidence interval [CI], 1.28 –1.74).24 Table 1 Summary of Studies on MAPT H1 Diplotype as Risk Factor for Parkinson‘s Disease The H1 and H2 haplotypes actually represent two distinct clades or families of subhaplotypes that arose from an inversion of 900kb on chromosome 17q21 approximately 3 million years ago.25 Since the event, the two inverted regions, which contain MAPT and several other genes, have been recombinationally suppressed and have accumulated sequence variation independently. As a result, any one of a large number of single nucleotide polymorphisms (SNPs) can be genotyped to differentiate the two haplotype clades (referred to hereafter as H1–H2 SNPs). However, because these SNPs are in perfect linkage disequilibrium (LD) with one another (r2 =1) across the entire region, the association of a given H1–H2 SNP with disease risk could be due to functional variation within any segment of MAPT or a neighboring gene.26 To more precisely map the disease-associated region in PD, Skipper and colleagues27 analyzed 14 SNPs specific to the H1 clade (ie, SNPs that were polymorphic on H1 chromosomes but monomorphic on H2 chromosomes) in subjects of the H1/H1 diplotype. A subhaplotype composed of two such “H1-SNPs” (rs242562 and rs2435207) spanning MAPT exons 1 to 4 was significantly overrepresented in cases versus control subjects refining the PD-associated interval from approximately 900 to 90kb. More comprehensive analyses of H1-SNPs have subsequently identified disease-associated H1 subhaplotypes overlapping the same MAPT region in PSP, CBD, and AD.28–30 In this study, we sought to confirm whether the H1 clade is associated with PD, and if so, whether the effect can be attributed to specific H1-SNPs or subhaplotypes previously implicated in PD and the tauopathies.

Objective Homozygous or compound heterozygous parkin mutations cause juvenile parkinsonism. Heterozygous parkin mutations are also found in patients with typical Parkinson's disease (PD), but it is unclear whether a single “mutation” in a patient is related to disease or is coincidental, because the mutation frequency in control subjects is unknown. We present a comprehensive sequence analysis of parkin in control subjects. Methods A total of 302 patients and 301 control subjects were sequenced, and findings were replicated in 1,260 additional patients and 1,657 control subjects. Results Thirty-four variants were detected, of which 21 were novel; 12 were polymorphisms and 22 were rare variants. Patients and control subjects did not differ in the frequency, type, or functional location of the variants. Even P437L, a common mutation thought to be pathogenic, was present in unaffected control subjects. Interpretation parkin point mutations are not exclusive to PD. The mere presence of a single point mutation in a patient, in the absence of a second mutation, should not be taken as a cause of disease unless corroborated by family data and functional studies. This study does not support the notion that heterozygous parkin sequence variants (mutations or polymorphisms) are risk factors for PD. Whether heterozygous dosage anomalies are associated with PD remains to be determined. Ann Neurol 2006

The G2019S mutation in the LRRK2 gene is reportedly a common cause of familial Parkinson's disease (PD) and may also have a significant role in nonfamilial PD. The objective of this study was to assess mutation carrier frequency in PD patients from movement disorder clinics in the United States, stratified by family history, age at onset, and geography; to determine carrier frequency in a large and well-characterized control population; to examine segregation of mutation in families of patients; and to correlate genotype with clinical phenotype. One thousand four hundred twenty-five unrelated PD patients from movement disorder clinics in Oregon, Washington, and New York and 1,647 unrelated controls were studied. The G2019S mutation was detected using a TaqMan assay and verified by sequencing. Eighteen of 1,425 patients and one of 1,647 controls had the mutation. Carrier frequency (± 2SE) in patients was 0.013 ± 0.006 overall, 0.030 ± 0.019 in familial PD, 0.007 ± 0.005 in nonfamilial PD, 0.016 ± 0.013 in early-onset PD, and 0.012 ± 0.007 in late-onset PD. Geographic differences were insignificant. Age at onset of mutation carriers ranged from 28 to 71 years. Mutation carriers were clinically indistinguishable from idiopathic PD. LRRK2 G2019S is the single most common pathogenic mutation linked to neurodegenerative disease to date. © 2005 Movement Disorder Society

Age-related variation in marker frequency can be a confounder in association studies, leading to both false positive and false negative findings and subsequently to inconsistent reproducibility. We have developed a simple method, based on a novel extension of moving average plots (MAP), which allows investigators to inspect the frequency data for hidden age-related variations. MAP uses the standard case-control association data and generates a birds-eye view of the frequency distributions across the age spectrum; a picture in which one can see if, how, and when the marker frequencies in cases differ from that in controls. The marker can be specified as an allele, genotype, haplotype, or environmental factor; and age can be age at onset, age when subject was last known to be unaffected, or duration of exposure. Signature patterns that emerge can help distinguish true disease associations from spurious associations due to age effects, age-varying associations from associations that are uniform across all ages, and associations with risk from associations with age-at-onset. Utility of MAP is illustrated by application to genetic and epidemiological association data for Alzheimer's and Parkinson's disease. MAP is intended as a descriptive method, to complement standard statistical techniques. Although originally developed for age patterns, MAP is equally useful for visualizing any quantitative trait.