Genome-Wide Analysis of nsLTP Gene Family and Identification of SiLTPs Contributing to High Oil Accumulation in Sesame (Sesamum indicum L.).

The biosynthesis and storage of lipids in oil crop seeds involve many gene families, such as nonspecific lipid-transfer proteins (nsLTPs). nsLTPs are cysteine-rich small basic proteins essential for plant development and survival. However, in sesame, information related to nsLTPs was limited. Thus, the objectives of this study were to identify the Sesamum indicum nsLTPs (SiLTPs) and reveal their potential role in oil accumulation in sesame seeds. Genome-wide analysis revealed 52 SiLTPs, nonrandomly distributed on 10 chromosomes in the sesame variety Zhongzhi 13. Following recent classification methods, the SiLTPs were divided into nine types, among which types I and XI were the dominants. We found that the SiLTPs could interact with several transcription factors, including APETALA2 (AP2), DNA binding with one finger (Dof), etc. Transcriptome analysis showed a tissue-specific expression of some SiLTP genes. By integrating the SiLTPs expression profiles and the weighted gene co-expression network analysis (WGCNA) results of two contrasting oil content sesame varieties, we identified SiLTPI.23 and SiLTPI.28 as the candidate genes for high oil content in sesame seeds. The presumed functions of the candidate gene were validated through overexpression of SiLTPI.23 in Arabidopsis thaliana. These findings expand our knowledge on nsLTPs in sesame and provide resources for functional studies and genetic improvement of oil content in sesame seeds.

Root diversity in sesame (Sesamum indicum L.): insights into the morphological, anatomical and gene expression profiles.

MAIN CONCLUSION: Sesame harbors a large diversity in root morphological and anatomical traits and a high root biomass improves the plant aboveground biomass as well as the seed yield. Sesame provides one of the most nutritious and healthy vegetable oils, sparking an increasing demand of its seeds. However, with the low yield and productivity of sesame, there is still a huge gap between the seed demand and supply. Improving the root system has a high potential to increase crop productivity, but information on the diversity of the sesame root systems is still lacking. In this study, 40 diverse sesame varieties were grown in soil and hydroponics systems and the diversity of the root system was investigated. The results showed that sesame holds a large root morphological and anatomical diversity, which can be harnessed in breeding programmes. Based on the clustering of the genotypes in hydroponics and soil culture systems, we found that similar genotypes were commonly clustered either in the small-root or in the big-root group, indicating that the hydroponics system can be employed for a large-scale root phenotyping. Our results further revealed that the root biomass positively contributes to increased seed yield in sesame, based on multi-environmental trials. By comparing the root transcriptome of two contrasting genotypes, 2897 differentially expressed genes were detected and they were enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, stilbenoid, diarylheptanoid and gingerol biosynthesis, flavonoid biosynthesis, suggesting that these pathways are crucial for sesame root growth and development. Overall, this study sheds light on the diversity of sesame root system and offers the basis for improving root traits and increasing sesame seed yield.

Robust identification of mosaic variants in congenital heart disease.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

Novel mutations in EVC cause aberrant splicing in Ellis-van Creveld syndrome.

Ellis-van Creveld syndrome (EvC) is a rare autosomal recessive disorder characterized by disproportionate chondrodysplasia, postaxial polydactyly, nail dystrophy, dental abnormalities and in a proportion of patients, congenital cardiac malformations. Weyers acrofacial dysostosis (Weyers) is another dominantly inherited disorder allelic to EvC syndrome but with milder phenotypes. Both disorders can result from loss-of-function mutations in either EVC or EVC2 gene, and phenotypes associated with the two gene mutations are clinically indistinguishable. We present here a clinical and molecular analysis of a Chinese family manifested specific features of EvC syndrome. Sequencing of both EVC and EVC2 identified two novel heterozygous splice site mutations c.384+5G>C in intron 3 and c.1465-1G>A in intron 10 in EVC, which were inherited from mother and father, respectively. In vitro minigene expression assay, RT-PCR and sequencing analysis demonstrated that c.384+5G>C mutation abolished normal splice site and created a new cryptic acceptor site within exon 4, whereas c.1465-1G>A mutation affected consensus splice junction site and resulted in full exon 11 skipping. These two aberrant pre-mRNA splicing processes both produced in-frame abnormal transcripts that possibly led to abolishment of important functional domains. To our knowledge, this is the first report of EVC mutations that cause EvC syndrome in Chinese population. Our data revealed that EVC splice site mutations altered splicing pattern and helped elucidate the pathogenesis of EvC syndrome.

Identification of rare variants in TNNI3 with atrial fibrillation in a Chinese GeneID population.

Despite advances by genome-wide association studies (GWAS), much of heritability of common human diseases remains missing, a phenomenon referred to as 'missing heritability'. One potential cause for 'missing heritability' is the rare susceptibility variants overlooked by GWAS. Atrial fibrillation (AF) is the most common arrhythmia seen at hospitals and increases risk of stroke by fivefold and doubles risk of heart failure and sudden death. Here, we studied one large Chinese family with AF and hypertrophic cardiomyopathy (HCM). Whole-exome sequencing analysis identified a mutation in TNNI3, R186Q, that co-segregated with the disease in the family, but did not exist in >1583 controls, suggesting that R186Q causes AF and HCM. High-resolution melting curve analysis and direct DNA sequence analysis were then used to screen mutations in all exons and exon-intron boundaries of TNNI3 in a panel of 1127 unrelated AF patients and 1583 non-AF subjects. Four novel missense variants were identified in TNNI3, including E64G, M154L, E187G and D196G in four independent AF patients, but no variant was found in 1583 non-AF subjects. All variants were not found in public databases, including the ExAC Browser database with 60,706 exomes. These data suggest that rare TNNI3 variants are associated with AF (P = 0.03). TNNI3 encodes troponin I, a key regulator of the contraction-relaxation function of cardiac muscle and was not previously implicated in AF. Thus, this study may identify a new biological pathway for the pathogenesis of AF and provides evidence to support the rare variant hypothesis for missing heritability.

Disease-associated variants in different categories of disease located in distinct regulatory elements.

BACKGROUND: The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. RESULTS: In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that regulatory regions are located within over 50% coding exon regions. Transcription promoters, methylation regions, and transcription insulators have the highest density of disease variants, with 472, 239, and 72 disease variants per one million base pairs, respectively. CONCLUSIONS: Disease-associated variants in different disease categories are preferentially located in particular regulatory elements. These results will be useful for an overall understanding about the differences among the pathogenic mechanisms of various disease-associated variants.

A PTPN11 allele encoding a catalytically impaired SHP2 protein in a patient with a Noonan syndrome phenotype.

The RASopathies are a relatively common group of phenotypically similar and genetically related autosomal dominant genetic syndromes caused by missense mutations affecting genes participating in the RAS/mitogen-activated protein kinase (MAPK) pathway that include Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML, formerly LEOPARD syndrome). NS and NSML can be difficult to differentiate during infancy, but the presence of multiple lentigines, café au lait spots, and specific cardiac defects facilitate the diagnosis. Furthermore, individual PTPN11 missense mutations are highly specific to each syndrome and engender opposite biochemical alterations on the function of SHP-2, the protein product of that gene. Here, we report on a 5-year-old male with two de novo PTPN11 mutations in cis, c.1471C>T (p.Pro491Ser), and c.1492C>T (p.Arg498Trp), which are associated with NS and NSML, respectively. This boy's phenotype is intermediate between NS and NSML with facial dysmorphism, short stature, mild global developmental delay, pulmonic stenosis, and deafness but absence of café au lait spots or lentigines. The double-mutant SHP-2 was found to be catalytically impaired. This raises the question of whether clinical differences between NS and NSML can be ascribed solely to the relative SHP-2 catalytic activity.

An algorithm to identify patients aged 0-3 with rare genetic disorders.

BACKGROUND: With over 7000 Mendelian disorders, identifying children with a specific rare genetic disorder diagnosis through structured electronic medical record data is challenging given incompleteness of records, inaccurate medical diagnosis coding, as well as heterogeneity in clinical symptoms and procedures for specific disorders. We sought to develop a digital phenotyping algorithm (PheIndex) using electronic medical records to identify children aged 0-3 diagnosed with genetic disorders or who present with illness with an increased risk for genetic disorders. RESULTS: Through expert opinion, we established 13 criteria for the algorithm and derived a score and a classification. The performance of each criterion and the classification were validated by chart review. PheIndex identified 1,088 children out of 93,154 live births who may be at an increased risk for genetic disorders. Chart review demonstrated that the algorithm achieved 90% sensitivity, 97% specificity, and 94% accuracy. CONCLUSIONS: The PheIndex algorithm can help identify when a rare genetic disorder may be present, alerting providers to consider ordering a diagnostic genetic test and/or referring a patient to a medical geneticist.

CADgene: a comprehensive database for coronary artery disease genes.

Coronary artery disease (CAD) is a complex, multifactorial disease and a leading cause of mortality world wide. Over the past decades, great efforts have been made to elucidate the underlying genetic basis of CAD and massive data have been accumulated. To integrate these data together and to provide a useful resource for researchers, we developed the CADgene, a comprehensive database for CAD genes. We manually extracted CAD-related evidence for more than 300 candidate genes for CAD from over 1300 publications of genetic studies. We classified these candidate genes into 12 functional categories based on their roles in CAD. For each gene, we extracted detailed information from related studies (e.g. the size of case-control, population, SNP, odds ratio, P-value, etc.) and made useful annotations, which include general gene information, Gene Ontology annotations, KEGG pathways, protein-protein interactions and others. Besides the statistical number of studies for each gene, CADgene also provides tools to search and show the most frequently studied candidate genes. In addition, CADgene provides cumulative data from 11 publications of CAD-related genome-wide association studies. CADgene has a user-friendly web interface with multiple browse and search functions. It is freely available at http://www.bioguo.org/CADgene/.

Significant association of SNP rs2106261 in the ZFHX3 gene with atrial fibrillation in a Chinese Han GeneID population.

Atrial fibrillation (AF) is the most common cardiac rhythm disorder at the clinical setting and accounts for up to 15% of all strokes. Recent genome-wide association studies (GWAS) identified two single nucleotide polymorphisms (SNPs), rs2106261 and rs7193343 in ZFHX3 (zinc finger homeobox 3 gene) and rs13376333 in KCNN3 (encoding a potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3) that showed significant association with AF in multiple populations of European ancestry. Here, we studied a Chinese Han, GeneID cohort consisting of 650 AF patients and 1,447 non-AF controls to test whether the GWAS findings on ZFHX3/KCNN3 and AF can be expanded to a different ethnic population. No significant association was detected for rs7193343 in ZFHX3 and rs13376333 in KCNN3. However, significant association was identified between rs2106261 in ZFHX3 and AF in the GeneID population for both allelic frequencies (P=0.001 after adjusting for covariates of age, gender, hypertension, coronary artery disease, and diabetes mellitus; OR=1.32), and genotypic frequencies assuming either an additive or recessive model (OR=1.29, P=0.001 and OR=1.77, P =0.00018, respectively). When only lone AF cases were analyzed, the association remained significant (OR=1.50, P=0.001 for allelic association; OR=1.45, P=0.001 for an additive model; OR=2.24, P=0.000043 for a recessive model). Our results indicate that rs2106261 in ZFHX3 confers a significant risk of AF in a Chinese Han population. The study expands the association between ZFHX3 and AF to a non-European ancestry population and provides the first evidence of a cross-race susceptibility of the 16q22 AF locus.

Development and Analytical Validation of a 29 Gene Clinical Pharmacogenetic Genotyping Panel: Multi-Ethnic Allele and Copy Number Variant Detection.

To develop a novel pharmacogenetic genotyping panel, a multidisciplinary team evaluated available evidence and selected 29 genes implicated in interindividual drug response variability, including 130 sequence variants and additional copy number variants (CNVs). Of the 29 genes, 11 had guidelines published by the Clinical Pharmacogenetics Implementation Consortium. Targeted genotyping and CNV interrogation were accomplished by multiplex single-base extension using the MassARRAY platform (Agena Biosciences) and multiplex ligation-dependent probe amplification (MRC Holland), respectively. Analytical validation of the panel was accomplished by a strategic combination of > 500 independent tests performed on 170 unique reference material DNA samples, which included sequence variant and CNV accuracy, reproducibility, and specimen (blood, saliva, and buccal swab) controls. Among the accuracy controls were 32 samples from the 1000 Genomes Project that were selected based on their enrichment of sequence variants included in the pharmacogenetic panel (VarCover.org). Coupled with publicly available samples from the Genetic Testing Reference Materials Coordination Program (GeT-RM), accuracy validation material was available for the majority (77%) of interrogated sequence variants (100% with average allele frequencies > 0.1%), as well as additional structural alleles with unique copy number signatures (e.g., CYP2D6*5, *13, *36, *68; CYP2B6*29; and CYP2C19*36). Accuracy and reproducibility for both genotyping and copy number were > 99.9%, indicating that the optimized panel platforms were precise and robust. Importantly, multi-ethnic allele frequencies of the interrogated variants indicate that the vast majority of the general population carries at least one of these clinically relevant pharmacogenetic variants, supporting the implementation of this panel for pharmacogenetic research and/or clinical implementation programs.

Deletion of ERF and CIC causes abnormal skull morphology and global developmental delay.

The ETS2 repressor factor (ERF) is a transcription factor in the RAS-MEK-ERK signal transduction cascade that regulates cell proliferation and differentiation, and pathogenic sequence variants in the ERF gene cause variable craniosynostosis inherited in an autosomal dominant pattern. The reported ERF variants are largely loss-of-function, implying haploinsufficiency as a primary disease mechanism; however, ERF gene deletions have not been reported previously. Here we describe three probands with macrocephaly, craniofacial dysmorphology, and global developmental delay. Clinical genetic testing for fragile X and other relevant sequencing panels were negative; however, chromosomal microarray identified heterozygous deletions (63.7-583.2 kb) on Chromosome 19q13.2 in each proband that together included five genes associated with Mendelian diseases (ATP1A3, ERF, CIC, MEGF8, and LIPE). Parental testing indicated that the aberrations were apparently de novo in two of the probands and were inherited in the one proband with the smallest deletion. Deletion of ERF is consistent with the reported loss-of-function ERF variants, prompting clinical copy-number-variant classifications of likely pathogenic. Moreover, the recent characterization of heterozygous loss-of-function CIC sequence variants as a cause of intellectual disability and neurodevelopmental disorders inherited in an autosomal dominant pattern is also consistent with the developmental delays and intellectual disabilities identified among the two probands with CIC deletions. Taken together, this case series adds to the previously reported patients with ERF and/or CIC sequence variants and supports haploinsufficiency of both genes as a mechanism for a variable syndromic cranial phenotype with developmental delays and intellectual disability inherited in an autosomal dominant pattern.

Minor allele C of chromosome 1p32 single nucleotide polymorphism rs11206510 confers risk of ischemic stroke in the Chinese Han population.

BACKGROUND AND PURPOSE: Genome-wide association studies found that the common allele T of single nucleotide polymorphism rs11206510 on chromosome 1p32 was associated with increased low-density lipoprotein-cholesterol levels (LDL-C) and with risk of coronary artery disease (CAD) in white populations. The goals of this study are to determine whether rs11206510 is associated with LDL-C and CAD in a different ethnic population, namely a Chinese cohort, and to investigate whether rs11206510 is associated with ischemic stroke. METHODS: The association of rs11206510 with LDL-C was analyzed in 1415 Chinese Han subjects. The CAD study utilized a GeneID cohort with 1543 CAD patients and 1240 controls. For stroke studies, 2 independent cohorts were used and included the GeneID North cohort, with 1205 cases and 1205 controls, and the GeneID Central cohort, with 692 cases and 882 controls. RESULTS: Different from white populations, the minor allele C of rs11206510 was associated with increased LDL-C levels in the Chinese Han population (adjusted P=0.002) and conferred risk of early-onset CAD (380 cases vs 1240 controls; adjusted P=0.002, odds ratio, 1.89), but not with overall CAD (adjusted P=0.82). The allelic association with ischemic stroke was highly significant in 2 independent cohorts, with adjusted P=1.13x10(-5) (odds ratio,1.71) in the GeneID North cohort and adjusted P=9.32x10(-5) (odds ratio, 1.70) in the GeneID Central cohort. Genotypic association was also significant for both early-onset CAD and ischemic stroke. CONCLUSIONS: Our results indicate that single nucleotide polymorphism rs11206510 is associated with LDL-C levels and early-onset CAD in the Chinese Han population. For the first time to our knowledge, this study also demonstrates that rs11206510 confers a significant risk of ischemic stroke.

Functional dominant-negative mutation of sodium channel subunit gene SCN3B associated with atrial fibrillation in a Chinese GeneID population.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the clinic, and accounts for more than 15% of strokes. Mutations in cardiac sodium channel alpha, beta1 and beta2 subunit genes (SCN5A, SCN1B, and SCN2B) have been identified in AF patients. We hypothesize that mutations in the sodium channel beta3 subunit gene SCN3B are also associated with AF. To test this hypothesis, we carried out a large scale sequencing analysis of all coding exons and exon-intron boundaries of SCN3B in 477 AF patients (28.5% lone AF) from the GeneID Chinese Han population. A novel A130V mutation was identified in a 46-year-old patient with lone AF, and the mutation was absent in 500 controls. Mutation A130V dramatically decreased the cardiac sodium current density when expressed in HEK293/Na(v)1.5 stable cell line, but did not have significant effect on kinetics of activation, inactivation, and channel recovery from inactivation. When co-expressed with wild type SCN3B, the A130V mutant SCN3B negated the function of wild type SCN3B, suggesting that A130V acts by a dominant negative mechanism. Western blot analysis with biotinylated plasma membrane protein extracts revealed that A130V did not affect cell surface expression of Na(v)1.5 or SCN3B, suggesting that mutant A130V SCN3B may not inhibit sodium channel trafficking, instead may affect conduction of sodium ions due to its malfunction as an integral component of the channel complex. This study identifies the first AF-associated mutation in SCN3B, and suggests that mutations in SCN3B may be a new pathogenic cause of AF.

[Genomewide association study: advances, challenges and deliberation].

Genomewide association study (GWAS) is a novel strategy for discovering genetic basis of human complex disease or trait. It utilizes millions of single nucleotide polymorphisms (SNPs) , which cover the whole genome, to conduct case-control association studies. In recent years, following the newly established Human Genome Project (HGP) and International Human HapMap project, large number of human complex disease/trait associated genetic variants have been identified through GWAS method, which provides important clues for understanding the mechanisms of related disorders. Due to the complexity of these multi-factorial diseases/traits and the complication of GWAS system itself, the GWAS remains many problems. In this review, we will discuss the potential issues in the following aspects as research methods, study subjects, genetic markers and statistical analysis.

Lessons learned from expanded reproductive carrier screening in self-reported Ashkenazi, Sephardi, and Mizrahi Jewish patients.

BACKGROUND: Next-generation sequencing (NGS)-based panels have gained traction as a strategy for reproductive carrier screening. Their value for screening Ashkenazi Jewish (AJ) individuals, who have benefited greatly from population-wide targeted testing, as well as Sephardi/Mizrahi Jewish (SMJ) individuals (an underserved population), has not been fully explored. METHODS: The clinical utilization by 6,805 self-reported Jewish individuals of an expanded NGS panel, along with several ancillary assays, was assessed retrospectively. Data were extracted for a subset of 96 diseases that, during the panel design phase, were classified as being AJ-, SMJ-, or pan-Jewish/pan-ethnic-relevant. RESULTS: 64.6% of individuals were identified as carriers of one or more of these 96 diseases. Over 80% of the reported variants would have been missed by following recommended AJ screening guidelines. 10.7% of variants reported for AJs were in "SMJ-relevant genes," and 31.2% reported for SMJs were in "AJ-relevant genes." Roughly 2.5% of individuals carried a novel, likely pathogenic variant. One in 16 linked cohort couples was identified as a carrier couple for at least one of these 96 diseases. CONCLUSION: For maximal carrier identification, this study supports using expanded NGS panels for individuals of all Jewish backgrounds. This approach can better empower at-risk couples for reproductive decision making.

Assessment of association of rs2200733 on chromosome 4q25 with atrial fibrillation and ischemic stroke in a Chinese Han population.

Atrial fibrillation (AF) is the most common arrhythmia in the clinical setting and an independent risk factor for stroke. Approximately 10 million Chinese people are affected by AF, but the genetic basis is largely unknown. A recent genome-wide association study in Iceland identified association between SNP rs2200733 on 4q25 and AF; however, many independent replication studies are essential to unequivocally validate this association. To assess the association between rs2200733 and AF as well as that between rs2200733 and ischemic stroke in a mainland Chinese Han population, we carried out case-control association studies with 383 AF patients versus 851 non-AF controls and 811 ischemic stroke patients versus 688 non-stroke controls. Highly significant association was detected between rs2200733 and AF in a Chinese Han population (allelic P = 3.7 × 10(-11) with OR = 1.81; genotypic P = 4.1 × 10(-12) with a dominant model). When the AF cases were divided into lone AF (32.6%) and other types of AF (67.4%), significantly stronger association was found with lone AF (OR = 2.40, P = 1.3 × 10(-9) compared to OR = 1.59, P = 6.2 × 10(-7) for other types of AF; P = 0.02 for two ORs). No significant association was found between rs2200733 and ischemic stroke. Our results suggest that SNP rs2200733 confers a highly significant risk of AF, but not ischemic stroke, in a more representative Chinese Han population in the mainland China.

Further evidence of genetic heterogeneity segregating with hereditary gingival fibromatosis.

AIM: To clinically characterize and map the disease-associated locus in a five-generation Chinese family with autosomal dominant early-onset hereditary gingival fibromatosis (HGF). MATERIAL AND METHODS: A complete oral examination was conducted. Genomic DNA samples were obtained from 14 individuals. Short tandem repeats markers, which encompass four previously known loci related to HGF, were genotyped. Two-point log of the odds (LOD) scores were calculated using MLINK program of the LINKAGE software, multipoint and non-parametric linkage (NPL) analysis were performed using the GENEHUNTER software. RESULTS: Clinical evaluation and histological examination of this family suggested typical features of HGF. The onset age was early in the generations, ranging between 1 and 2 years. None of the tested markers showed cosegregation among affected individuals. Genotyping data from four putative regions yielded significant negative two-point LOD scores (<-2.0) at theta=0. The maximum multipoint LOD scores and NPL analysis revealed exclusion of these loci as well. CONCLUSIONS: Exclusion of linkage in this family to any of the known HGF loci proved the existence of a novel locus for autosomal dominant HGF and showed that this rare disorder is far more heterogeneous than previously expected.

Prenatal cytogenomic identification and molecular refinement of compound heterozygous STRC deletion breakpoints.

Here, we report the prenatal detection of a compound heterozygous deletion at chromosome 15q15.3 by clinical chromosomal microarray (CMA) testing that included the CATSPER2 male infertility gene. However, given the low resolution of CMA at this homologous locus, it was unclear if the neighboring STRC hearing loss gene was also affected. Therefore, we developed a novel allele-specific PCR strategy, which narrowed the proximal breakpoint of the maternally inherited deletion to a 310 bp interval that was 440 bp upstream from the STRC transcription start site.

Integrated CYP2D6 interrogation for multiethnic copy number and tandem allele detection.

AIM: To comprehensively interrogate CYP2D6 by integrating genotyping, copy number analysis and novel strategies to identify CYP2D6*36 and characterize CYP2D6 duplications. METHODS: Genotyping of 16 CYP2D6 alleles, multiplex ligation-dependent probe amplification (MLPA) and CYP2D6*36 and duplication allele-specific genotyping were performed on 427 African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals. RESULTS: A novel PCR strategy determined that almost half of all CYP2D6*10 (100C>T) alleles are actually *36 (isolated or in tandem with *10) and all identified duplication alleles were characterized. Integrated results from all testing platforms enabled the refinement of genotype frequencies across all studied populations. CONCLUSION: The polymorphic CYP2D6 gene requires comprehensive interrogation to characterize allelic variation across ethnicities, which was enabled in this study by integrating multiplexed genotyping, MLPA copy number analysis, novel PCR strategies and duplication allele-specific genotyping.