Comprehensive population screening in the Ashkenazi Jewish population for recurrent disease-causing variants.

The Ashkenazi Jewish (AJ) population has an increased risk for a variety of recessive diseases due to historical founder effects and genetic drift. For some, the disease-causing founder mutations have been identified and well-characterized, but for others, further study is necessary. The purpose of this study is to assess the carrier frequencies of 85 pathogenic variants causative of 29 recessive conditions in the AJ population. Up to 3000 AJ individuals were genotyped by Luminex MagPlex® -TAG™ bead array or Agena Bioscience™ MassARRAY assays. We identified seven conditions with carrier frequencies higher than 1 in 100, nine between 1 in 100 and 1 in 200, and four between 1 in 200 and 1 in 500. Variants in nine conditions had a detected carrier rate of less than 1 in 500 or were not identified in approximately 2000 AJ individuals. We assessed the combined AJ carrier frequency for 18 relatively prevalent diseases to be 1 in 6, and the risk of AJ individuals to be a carrier couple for one of these 18 diseases as 1 in 441. We note additional recessive genetic conditions should be considered for AJ carrier screening panels.

Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population.

Hoyeraal-Hreidarsson syndrome (HH) is a clinically severe variant of dyskeratosis congenita (DC), characterized by cerebellar hypoplasia, microcephaly, intrauterine growth retardation, and severe immunodeficiency in addition to features of DC. Germline mutations in the RTEL1 gene have recently been identified as causative of HH. In this study, the carrier frequency for five RTEL1 mutations that occurred in individuals of Ashkenazi Jewish descent was investigated in order to advise on including them in existing clinical mutation panels for this population. Our screening showed that the carrier frequency for c.3791G>A (p.R1264H) was higher than expected, 1% in the Ashkenazi Orthodox and 0.45% in the general Ashkenazi Jewish population. Haplotype analyses suggested the presence of a common founder. We recommend that the c.3791G>A RTEL1 mutation be considered for inclusion in carrier screening panels in the Ashkenazi population.

Cognitive and Antipsychotic Medication Use in Monoallelic GBA-Related Parkinson Disease.

Mutations in glucosidase, beta, acid (GBA) are associated with cognitive impairment in Parkinson disease (PD) as well as dementia with Lewy bodies. For both of these diseases, dementia and hallucinations are typically treated with cholinesterase inhibitors and antipsychotics. However, in some lysosomal storage disorders certain antipsychotic medications are poorly tolerated. This study examined cholinesterase inhibitor and antipsychotic use in monoallelic GBA-related PD to explore potential pharmacogenetic relationships. Monoallelic GBA mutation carriers with PD (GBA-PD) with at least two clinic visits (n = 34) were matched for age-of-onset and gender to GBA and leucine-rich repeat kinase 2 (LRRK2) mutation negative idiopathic PD subjects (IPD) (n = 60). Information regarding cholinesterase inhibitor and antipsychotic use as well as impaired cognition (UPDRS Mentation >1) and hallucinations (UPDRS Thought Disorder >1) were obtained. GBA-PD more frequently reported hallucinations (HR = 5.0; p = 0.01) and they were more likely to have cognitive impairment but this was not statistically significant (HR 2.2, p = 0.07). Antipsychotic use was not significantly different between GBA-PD and IPD (HR = 1.9; p = 0.28), but GBA-PD were more likely to have sustained cholinesterase inhibitor use (HR = 3.1; p = 0.008), even after adjustment for cognition and hallucinations. Consistent with reports of worse cognition, GBA-PD patients are more likely to use cholinesterase inhibitors compared to IPD. While there was no difference in antipsychotic use between IPD and GBA-PD, persistent use of quetiapine in GBA-PD suggests that it is tolerated and that a significant interaction is unlikely. Further prospective study in larger samples with more extensive cognitive assessment is warranted to better understand pharmacogenetic relationships in GBA-PD.

A founder mutation in COL4A3 causes autosomal recessive Alport syndrome in the Ashkenazi Jewish population.

Alport syndrome is an inherited progressive nephropathy arising from mutations in the type IV collagen genes, COL4A3, COL4A4, and COL4A5. Symptoms also include sensorineural hearing loss and ocular lesions. We determined the molecular basis of Alport syndrome in a non-consanguineous Ashkenazi Jewish family with multiple affected females using linkage analysis and next generation sequencing. We identified a homozygous COL4A3 mutation, c.40_63del, in affected individuals with mutant alleles inherited from each parent on partially conserved haplotypes. Large-scale population screening of 2017 unrelated Ashkenazi Jewish samples revealed a carrier frequency of 1 in 183 indicating that COL4A3 c.40_63del is a founder mutation which may be a common cause of Alport syndrome in this population. Additionally, we determined that heterozygous mutation carriers in this family do not meet criteria for a diagnosis of Thin Basement Membrane Nephropathy and concluded that carriers of c.40_63del are not likely to develop benign familial hematuria.

Multi-ethnic distribution of clinically relevant CYP2C genotypes and haplotypes.

To determine CYP2C19 and CYP2C8 allele frequencies, 28 coding and/or functional variants were genotyped in 1250 African-American, Asian, Caucasian, Hispanic and Ashkenazi Jewish (AJ) individuals. The combined CYP2C19 variant allele frequencies ranged from ∼0.30 to 0.41; however, the CYP2C8 frequencies were much lower (∼0.04-0.13). After incorporating previously reported CYP2C9 genotyping results from these populations (36 total CYP2C variants), 16 multi-ethnic CYP2C haplotypes were inferred with frequencies >0.5%. Notably, the 2C19*17-2C9*1-2C8*2 haplotype was identified among African-Americans (8%) and Hispanics (2%), indicating that CYP2C19*17 does not always tag a CYP2C haplotype that encodes efficient CYP2C-substrate metabolism. The 2C19*1-2C9*2-2C8*3 haplotype was identified in all populations except African-Americans and additional novel haplotypes were identified in selected populations (for example, 2C19*2-2C9*1-2C8*4 and 2C19*4B-2C9*1-2C8*1), together indicating that both CYP2C19*17 and *2 can be linked with other CYP2C loss-of-function alleles. These results have important implications for pharmacogenomic association studies involving the CYP2C locus and are clinically relevant when administering CYP2C-substrate medications.

Identification of CYP2C19*4B: pharmacogenetic implications for drug metabolism including clopidogrel responsiveness.

CYP2C19 is a principal enzyme involved in the bioactivation of the antiplatelet prodrug clopidogrel and common CYP2C19 loss-of-function alleles are associated with adverse cardiovascular events. To assess the impact of the CYP2C19*17 increased activity allele in the Ashkenazi Jewish (AJ) and Sephardi Jewish (SJ) populations and to determine the frequencies of additional variant alleles, 250 AJ and 135 SJ individuals were genotyped for CYP2C19*2-*10, *12-*17, *22 and P-glycoprotein (ABCB1) c.3435C>T. Importantly, CYP2C19*4, a loss-of-function allele, was identified in linkage disequilibrium with *17. This novel haplotype, designated CYP2C19*4B, significantly alters the interpretation of CYP2C19 genotyping when testing *17. Moreover, genotyping CYP2C19*17 changed the frequency of extensive metabolizers from ∼70 to ∼40%, reclassifying ∼30% as ultrarapid metabolizers. Combining CYP2C19 and ABCB1 identified ∼1 in 3 AJ and ∼1 in 2 SJ individuals at increased risk for adverse responses to clopidogrel. These data underscore the importance of including *4B and *17 when clinically genotyping CYP2C19.

PTPN11 analysis for the prenatal diagnosis of Noonan syndrome in fetuses with abnormal ultrasound findings.

Noonan syndrome (NS) is an autosomal dominant disorder characterized by short stature, congenital heart defects and distinctive facies. The disorder is genetically heterogeneous with approximately 50% of patients having PTPN11 mutations. Prenatally, the diagnosis of NS has been suspected following certain ultrasound findings, such as cystic hygroma, increased nuchal translucency (NT) and hydrops fetalis. Studies of fetuses with cystic hygroma have suggested an NS prevalence of 1-3%. A retrospective review was performed to assess the utility of PTPN11 testing based on prenatal sonographic findings (n = 134). The most commonly reported indications for testing were increased NT and cystic hygroma. Analysis showed heterozygous missense mutations in 12 fetuses, corresponding to a positive test rate of 9%. PTPN11 mutations were identified in 16% and 2% of fetuses with cystic hygroma and increased NT, respectively. Among fetuses with isolated cystic hygroma, PTPN11 mutation prevalence was 11%. The mutations observed in the three fetuses with hydrops fetalis had previously been reported as somatic cancer mutations. Prenatal PTPN11 testing has diagnostic and possible prognostic properties that can aid in risk assessment and genetic counseling. As NS is genetically heterogeneous, negative PTPN11 testing cannot exclude the diagnosis and further study is warranted regarding the other NS genes.

Prenatal genetic screening in the Ashkenazi Jewish population.

The Ashkenazi Jewish community is a unique and ideal population in which to provide multiple disease screening because detection rates are high (> 95%) by testing a limited number of mutations. The residual risk that remains is very low. In addition, the lessons learned from carrier screening in this community indicate that only through genetic counseling and education can screening in the general population gain wide acceptance and provide maximum benefit.

Maple syrup urine disease: identification and carrier-frequency determination of a novel founder mutation in the Ashkenazi Jewish population.

Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid metabolism. We noted that a large proportion (10 of 34) of families with MSUD that were followed in our clinic were of Ashkenazi Jewish (AJ) descent, leading us to search for a common mutation within this group. On the basis of genotyping data suggestive of a conserved haplotype at tightly linked markers on chromosome 6q14, the BCKDHB gene encoding the E1beta subunit was sequenced. Three novel mutations were identified in seven unrelated AJ patients with MSUD. The locations of the affected residues in the crystal structure of the E1beta subunit suggested possible mechanisms for the deleterious effects of these mutations. Large-scale population screening of AJ individuals for R183P, the mutation present in six of seven patients, revealed that the carrier frequency of the mutant allele was approximately 1/113; the patient not carrying R183P had a previously described homozygous mutation in the gene encoding the E2 subunit. These findings suggested that a limited number of mutations might underlie MSUD in the AJ population, potentially facilitating prenatal diagnosis and carrier detection of MSUD in this group.

Nonradioactive detection of the common Connexin 26 167delT and 35delG mutations and frequencies among Ashkenazi Jews.

Mutations in the gap junction beta2 (GJB2) gene, Connexin 26 (Cx26), cause nonsyndromic sensorineural recessive deafness (NSRD). Two frameshift mutations, 167delT and 35delG, are the most frequent Cx26 lesions causing NSRD. The 35delG mutation is panethnic, while the 167delT lesion occurs almost exclusively in the Ashkenazi Jewish population at a carrier frequency of 2 to 4%. To facilitate carrier detection, a simple nonradioactive allele-specific oligonucleotide (ASO) hybridization assay was developed for the 167delT and 35delG mutations. Screening of 1012 anonymous Ashkenazi Jewish individuals from the New York Metropolitan area revealed carrier frequencies for 167delT and 35delG of 3.96% (95% CI: 2.75-5.15%) and 0.69% (95% CI: 0.18-1.20%), respectively. This sensitive, specific, and relatively inexpensive method can reliably identify affected newborns and patients with NSRD as well as facilitate carrier screening for Connexin 26 deafness in the Ashkenazi Jewish community.

Association between serum triiodothyronine (t3) level and risk of disease recurrence in men with localized prostate cancer.

We assessed the relationship of serum triiodothyronine (t3) level and risk of disease recurrence in men treated for localized prostate cancer. Participants in our study were found through urology and radiation oncology clinics, and all eligible patients were asked to take part. All patients had been initially diagnosed on the basis of rising prostate specific antigen (PSA) or abnormal physical examination. Histological confirmation of diagnosis was obtained for all subjects. Serum (t3) level was determined by chemoluminescent assay with a standard, commercially available instrument (Immulite Diagnostic Products Corporation, Los Angeles, California). Sixty-eight men with prostate cancer were studied. In our treatment protocol, patients are divided into three risk groups: low risk: serum PSA7, tumor in seminal vesicle biopsy, serum PSA >15 or stage T2c or T3. These patients are treated with 3 months combined hormonal therapy, an implant, and after 2 months break 6000 rad external beam radiotherapy. There was a significant increase in serum t3 with risk category (P=0.011). Tukey's multiple range B-test showed a significant difference between the t3 levels of the high risk patients, when compared to the t3 levels of the moderate (P=0.013) and low risk patients (P=0.041). The range test showed no significant difference between the t3 levels of the moderate and low risk patients (P=0.897). Because t3 levels may be affected by age, we performed multivariate linear regression, with t3 as the dependent variable. There was a statistically significant (P=0.035) association of t3 level with risk group, but there was no significant association of t3 with age (P=0.803). Multivariate linear regression, with t3 as dependent variable, PSA, Gleason score, and stage as independent variables showed a significant overall association of the three independent variables with t3 (P=0.042), though individually the relationships were not significant. None of the men had a t3 level that was above the normal range for our laboratory (137 ng/dl). Urologists are actively seeking additional biomarkers of prostate cancer aggressiveness. Many prostate cancers are quite indolent and may never cause a problem, but it is impossible to identify such tumors with certainty. Further studies of serum t3 level as a biomarker in prostate cancer might therefore be worthwhile. With more and better biomarkers, many older men might be spared the rigors of radiation therapy and/or surgery and the complications. Also, new prostate cancer therapies might be directed toward inhibiting the mitogenic effects of t3.Prostate Cancer and Prostatic Diseases (2001) 4, 232-234.

Body mass, age, and the APC I1307K allele in Ashkenazi Jewish prostate cancer patients.

The I1307K mutation of the adenopolyposis coli gene (APC), located on chromosome 5q21-q22, is associated with an increased risk of cancer in Ashkenazi Jews. In the present study, we analyzed age and body mass of Ashkenazi Jewish prostate cancer patients, with and without the APC I1307K mutation. Participants in our study were found through urology and radiation oncology clinics, and all eligible patients were asked to take part. A familial history was obtained by interview or self-report questionnaire. Histological confirmation of diagnosis was obtained for all subjects. The I1307K allele of the APC gene was detected by amplification of lymphocyte DNA from peripheral blood according to standard polymerase chain reaction (PCR) and dot blot procedures. We studied 135 Ashkenazi Jewish men with prostate cancer. The youngest was 49, the oldest 80, average age 68 +/- 6.88 (mean +/- SD). The older patients carrying the wild type APC allele tended to have a lower body mass than the younger ones (r = -.27, P =.002). Of 71 patients under 70 years old, 65 carried the wild type APC allele, and had a body mass index of 28. 7 +/- 4.23 kg/m(2). The six men under age 70 carrying the I1307K APC allele had a body mass index of 26.87 +/- 1.44 kg/m(2). The difference in body mass index of the two groups is significant (P =. 032, t test for unequal variance). Increased body mass is a prostate cancer risk factor, and hereditary prostate cancer is associated with younger patients. Therefore, our finding, that patients under age 70 carrying the I1307K allele are significantly thinner than those carrying the wild type allele, suggests that the APC I1307K allele is also a prostate cancer risk factor. Our results are in accord with other studies indicating that APC mutations increase the risk of prostate cancer.

CAG expansion affects the expression of mutant Huntingtin in the Huntington's disease brain.

A trinucleotide repeat (CAG) expansion in the huntingtin gene causes Huntington's disease (HD). In brain tissue from HD heterozygotes with adult onset and more clinically severe juvenile onset, where the largest expansions occur, a mutant protein of equivalent intensity to wild-type huntingtin was detected in cortical synaptosomes, indicating that a mutant species is synthesized and transported with the normal protein to nerve endings. The increased size of mutant huntingtin relative to the wild type was highly correlated with CAG repeat expansion, thereby linking an altered electrophoretic mobility of the mutant protein to its abnormal function. Mutant huntingtin appeared in gray and white matter with no difference in expression in affected regions. The mutant protein was broader than the wild type and in 6 of 11 juvenile cases resolved as a complex of bands, consistent with evidence at the DNA level for somatic mosaicism. Thus, HD pathogenesis results from a gain of function by an aberrant protein that is widely expressed in brain and is harmful only to some neurons.

Fabry disease: detection of gene rearrangements in the human alpha-galactosidase A gene by multiplex PCR amplification.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from lesions in the alpha-galactosidase A gene leading to deficient or absent activity of the lysosomal hydrolase. To facilitate the detection of rearrangements in this 14-kb gene, a method was developed for the PCR amplification of all seven exons from genomic DNA in a single multiplex reaction. The entire coding region and all the intron/exon boundaries were amplified as four products. Application of this method permitted the detection of all five partial deletions previously identified by Southern analysis. This rapid method can be used to identify gene rearrangements in affected hemizygotes and determine heterozygosity for at risk females in families with Fabry disease.

Amplification of human polymorphic sites in the X-chromosomal region q21.33 to q24: DXS17, DXS87, DXS287, and alpha-galactosidase A.

Methods for the PCR amplification of five polymorphic sites in the region Xq21.33 to Xq24 were developed and used to predict heterozygosity for Fabry disease in informative families. Clones containing polymorphic sites associated with DNA segments DXS17, DXS87, and DXS287, and the alpha-galactosidase A gene were isolated from genomic libraries. Surrounding nucleotide sequences and optimal conditions for amplification of each polymorphic site were determined. These amplifiable polymorphisms provided predictions of heterozygosity for Fabry disease and should be useful for diagnostic linkage analyses in Alport syndrome, X-linked cleft palate and ankyloglossia, Pelizaeus-Merzbacher disease, and X-linked agammaglobulinemia as well as sequence-tagged sites for gene mapping.

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene.

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Southern hybridization analysis of the alpha-galactosidase gene in affected hemizygous males from 130 unrelated families with Fabry disease revealed six with different gene rearrangements and one with an exonic point mutation resulting in the obliteration of an Msp I restriction site. Five partial gene deletions were detected ranging in size from 0.4 to greater than 5.5 kb. Four of these deletions had breakpoints in intron 2, a region in the gene containing multiple Alu repeat sequences. A sixth genomic rearrangement was identified in which a region of about 8 kb, containing exons 2 through 6, was duplicated by a homologous, but unequal crossover event. The Msp I site obliteration, which mapped to exon 7, was detected in an affected hemizygote who had residual enzyme activity. Genomic amplification by the polymerase chain reaction and sequencing revealed that the obliteration resulted from a C to T transition at nucleotide 1066 in the coding sequence. This point mutation, the first identified in Fabry disease, resulted in an arginine356 to tryptophan356 substitution which altered the enzyme's kinetic and stability properties. The detection of these abnormalities provided for the precise identification of Fabry heterozygotes, thereby permitting molecular pedigree analysis in these families which revealed paternity exclusions and the first documented new mutations in this disease.