Validation and application of a novel integrated genetic screening method to a cohort of 1,112 men with idiopathic azoospermia or severe oligozoospermia.

Microdeletions of the Y chromosome (YCMs), Klinefelter syndrome (47,XXY), and CFTR mutations are known genetic causes of severe male infertility, but the majority of cases remain idiopathic. Here, we describe a novel method using single molecule Molecular Inversion Probes (smMIPs), to screen infertile men for mutations and copy number variations affecting known disease genes. We designed a set of 4,525 smMIPs targeting the coding regions of causal (n = 6) and candidate (n = 101) male infertility genes. After extensive validation, we screened 1,112 idiopathic infertile men with non-obstructive azoospermia or severe oligozoospermia. In addition to five chromosome YCMs and six other sex chromosomal anomalies, we identified five patients with rare recessive mutations in CFTR as well as a patient with a rare heterozygous frameshift mutation in SYCP3 that may be of clinical relevance. This results in a genetic diagnosis in 11-17 patients (1%-1.5%), a yield that may increase significantly when more genes are confidently linked to male infertility. In conclusion, we developed a flexible and scalable method to reliably detect genetic causes of male infertility. The assay consolidates the detection of different types of genetic variation while increasing the diagnostic yield and detection precision at the same or lower price compared with currently used methods.

DeNovoGear: de novo indel and point mutation discovery and phasing.

We present DeNovoGear software for analyzing de novo mutations from familial and somatic tissue sequencing data. DeNovoGear uses likelihood-based error modeling to reduce the false positive rate of mutation discovery in exome analysis and fragment information to identify the parental origin of germ-line mutations. We used DeNovoGear on human whole-genome sequencing data to produce a set of predicted de novo insertion and/or deletion (indel) mutations with a 95% validation rate.

Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1.

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man's risk of disease by 10% (OR 1.10 [1.04-1.16], p<2 × 10(-3)), rare X-linked CNVs by 29%, (OR 1.29 [1.11-1.50], p<1 × 10(-3)), and rare Y-linked duplications by 88% (OR 1.88 [1.13-3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2 × 10(-5)). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.

Validating single-cell genomics for the study of renal development.

Single-cell genomics will enable studies of the earliest events in kidney development, although it is unclear if existing technologies are mature enough to generate accurate and reproducible data on kidney progenitors. Here we designed a pilot study to validate a high-throughput assay to measure the expression levels of key regulators of kidney development in single cells isolated from embryonic mice. Our experiment produced 4608 expression measurements of 22 genes, made in small cell pools, and 28 single cells purified from the RET-positive ureteric bud. There were remarkable levels of concordance with expression data generated by traditional microarray analysis on bulk ureteric bud tissue with the correlation between our average single-cell measurements and GUDMAP measurements for each gene of 0.82-0.85. Nonetheless, a major motivation for single-cell technology is to uncover dynamic biology hidden in population means. There was evidence for extensive and surprising variation in expression of Wnt11 and Etv5, both downstream targets of activated RET. The variation for all genes in the study was strongly consistent with burst-like promoter kinetics. Thus, our results can inform the design of future single-cell experiments, which are poised to provide important insights into kidney development and disease.

Intrachromosomal homologous recombination between inverted amplicons on opposing Y-chromosome arms.

Amplicons--large, nearly identical repeats in direct or inverted orientation--are abundant in the male-specific region of the human Y chromosome (MSY) and provide targets for intrachromosomal non-allelic homologous recombination (NAHR). Thus far, NAHR events resulting in deletions, duplications, inversions, or isodicentric chromosomes have been reported only for amplicon pairs located exclusively on the short arm (Yp) or the long arm (Yq). Here we report our finding of four men with Y chromosomes that evidently formed by intrachromosomal NAHR between inverted repeat pairs comprising one amplicon on Yp and one amplicon on Yq. In two men with spermatogenic failure, sister-chromatid crossing-over resulted in pseudoisoYp chromosome formation and loss of distal Yq. In two men with normal spermatogenesis, intrachromatid crossing-over generated pericentric inversions. These findings highlight the recombinogenic nature of the MSY, as intrachromosomal NAHR occurs for nearly all Y-chromosome amplicon pairs, even those located on opposing chromosome arms.

Gene copy number reduction in the azoospermia factor c (AZFc) region and its effect on total motile sperm count.

The azoospermia factor c (AZFc) region harbors multi-copy genes that are expressed in the testis. Deletions of the AZFc region lead to reduced copy numbers of these genes. Four (partial) AZFc deletions have been described of which the b2/b4 and gr/gr deletions affect semen quality. In most studies, (partial) AZFc deletions are identified and characterized using plus/minus sequence site tag (STS) polymerase chain reaction (PCR). However, secondary duplications increase the gene copy number without re-introducing the STS boundary marker. Consequently, the actual copy number of AZFc genes cannot be determined via STS PCR. In the current study, we first set out to determine by quantitative real-time PCR the actual copy number of all AZFc genes in men with (partial) AZFc deletions based on STS PCR. We then analyzed whether reduced gene copy numbers of each AZFc gene family were associated with reduced total motile sperm count (TMC), regardless of the type of deletion. We screened 840 men and identified 31 unrelated men with (partial) deletions of AZFc based on STS PCR. Of these 31 men, 6 men (19%) had one or more secondary duplications. For all AZFc genes, we found an association between a reduction in the copy number of each individual AZFc gene and reduced TMC. In gr/gr-deleted men, restoration of reduced gene copy numbers restored their TMC to normal values. Our findings suggest that the gene content of the AZFc region has been preserved throughout evolution through a dosage effect of the AZFc genes on TMC safeguarding male fertility.

The origins and functional effects of postzygotic mutations throughout the human life span.

Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remain unclear. To study the origins and functional consequences of PZMs, we generated a multitissue atlas of PZMs spanning 54 tissue and cell types from 948 donors. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, and 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we found that their type and predicted functional impact vary during prenatal development, across tissues, and through the germ cell life cycle. Thus, methods for interpreting effects across the body and the life span are needed to fully understand the consequences of genetic variants.

The human Y chromosome: a masculine chromosome.

Once considered to be a genetic wasteland of no scientific interest beyond sex determination, the human Y chromosome has made a significant comeback in the past few decades and is currently implicated in multiple diseases, including spermatogenic failure - absent or very low levels of sperm production. The Y chromosome contains over one hundred testis-specific transcripts, and several deletions have been described that remove some of these transcripts, thereby causing spermatogenic failure. Screening for such deletions in infertile men is now a standard part of clinical evaluation. Many other Y-chromosome structural variants, some of which affect gene copy number, have been reported recently, and future research will be necessary to address the phenotypic effect of these structural variants.

Y chromosome TSPY copy numbers and semen quality.

OBJECTIVE: To determine whether variation in testis-specific protein Y-encoded (TSPY) gene copy number affects semen quality. DESIGN: Nested case-control study. SETTING: University hospital. PATIENT(S): From a consecutive cohort of 1,016 male partners of subfertile couples, unselected for sperm counts, we selected as cases 100 men with the lowest total number of progressively motile sperm (TMC) and as controls, 100 men with the highest total number of progressively motile sperm. INTERVENTION(S): Quantitative real-time polymerase chain reaction (PCR) and Southern blot to determine TSPY copy number. MAIN OUTCOME MEASURE(S): TSPY copy number. RESULT(S): The quantitative PCR method showed excellent agreement with the Southern blot analysis. Cases had a median TSPY copy number of 35 (range 20-73), whereas controls had a median TSPY copy number of 34 (range 26-76). This difference was not statistically significant. CONCLUSION(S): We found no association between TSPY copy numbers and severe spermatogenic failure. The observed variation in TSPY copy number therefore appears to have no functional consequences for semen quality.

Ligand selectivity of gonadotropin receptors. Role of the beta-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor.

The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.