Pierre-Simon Jouk

Homozygous mutation of AURKC yields large-headed polyploid spermatozoa and causes male infertility

The World Health Organization conservatively estimates that 80 million people suffer from infertility worldwide. Male factors are believed to be responsible for 20–50% of all infertility cases, but microdeletions of the Y chromosome are the only genetic defects altering human spermatogenesis that have been reported repeatedly. We focused our work on infertile men with a normal somatic karyotype but typical spermatozoa mainly characterized by large heads, a variable number of tails and an increased chromosomal content (OMIM 243060). We performed a genome-wide microsatellite scan on ten infertile men presenting this characteristic phenotype. In all of these men, we identified a common region of homozygosity harboring the aurora kinase C gene (AURKC) with a single nucleotide deletion in the AURKC coding sequence. In addition, we show that this founder mutation results in premature termination of translation, yielding a truncated protein that lacks the kinase domain. We conclude that the absence of AURKC causes male infertility owing to the production of large-headed multiflagellar polyploid spermatozoa.

Mutations in DNAH1, which Encodes an Inner Arm Heavy Chain Dynein, Lead to Male Infertility from Multiple Morphological Abnormalities of the Sperm Flagella

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia

STUDY QUESTIONnDo DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia?nnnSUMMARY ANSWERnTwo DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion.nnnWHAT IS KNOWN ALREADYnGlobozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing.nnnSTUDY DESIGN, SIZE, DURATIONnIn this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWe developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThere were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients.nnnLIMITATIONS, REASONS FOR CAUTIONnGlobally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups.nnnWIDER IMPLICATIONS OF THE FINDINGSnIn previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background.nnnSTUDY FUNDING/COMPETING INTEREST(S)nNone of the authors have any competing interest. This work is part of the project Identification and Characterization of Genes Involved in Infertility (ICG2I) funded by the program GENOPAT 2009 from the French Research Agency (ANR).

Method for the study of the three‐dimensional orientation of the nuclei of myocardial cells in fetal human heart by means of confocal scanning laser microscopy

A series of three‐dimensional image analysis tools are used to measure the three‐dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 μm inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.

17p13.1 microduplication in a boy with Silver-Russell syndrome features and intellectual disability

Many deletions of chromosome 17p13.1 have been described, but very few 17p13.1 duplications have been reported yet. Here, we describe the genotype and phenotype of a boy with a duplication of this region. The main clinical features are mild intellectual deficiency, growth retardation, and a typical Silver–Russell syndrome (SRS) appearance with small triangular face, prominent forehead, micrognathia, low‐set ears, and clinodactyly. Array‐CGH revealed a 586u2009kb duplication containing many genes with a high neuronal expression. Interestingly, this region covers the minimal critical region including all candidate genes suggested to explain the 17p13.1 microdeletion syndrome. In the neighboring region 17p13.3, deletions and duplications of the same region are each responsible of a specific phenotype. Future case descriptions will show if a similar mechanism applies to the region 17p13.1. The 17p13.1 region contains interesting putative candidate genes that might be involved in the SRS etiology. Additional data are needed to verify the significance of this aberration.

Low but Increasing Prevalence of Autism Spectrum Disorders in a French Area from Register-Based Data

AbstractRegister-based prevalence rates of childhood autism (CA), Asperger’s syndrome (AS) and other autism spectrum disorders (ASD) were calculated among children aged 7xa0years old of the 1997–2003 birth cohorts, living in four counties in France. The proportion of children presenting comorbidities was reported. 1123 children with ASD were recorded (M/F ratio: 4.1), representing an overall prevalence rate of 36.5/10,000 children (95xa0% CI 34.4–38.7): 8.8/10,000 for CA (95xa0% CI 7.8–9.9), 1.7/10,000 for AS (95xa0% CI 1.3–2.3) and 25.9/10,000 for other ASD (95xa0% CI 24.2–27.8). ASD prevalence significantly increased (pxa0<xa00.0001) during the period under study. The proportion of children with an intellectual disability was 47.3xa0%, all other comorbidities were present in less than 5xa0% of the cases.n

Stillbirth classification in population-based data and role of fetal growth restriction: the example of RECODE

BackgroundStillbirth classifications use various strategies to synthesise information associated with fetal demise with the aim of identifying key causes for the death. RECODE is a hierarchical classification of death-related conditions, which grants a major place to fetal growth restriction (FGR). Our objective was to explore how placement of FGR in the hierarchy affected results from the classification.MethodsIn the Rhône-Alpes region, all stillbirths were recorded in a local registry from 2000 to 2010 in three districts (Nu2009=u2009969). Small for gestational age (SGA) was defined as a birthweight below the 10th percentile. We applied RECODE and then modified the hierarchy, including FGR as the penultimate category (RECODE-R).Results49.0% of stillbirths were SGA. From RECODE to RECODE-R, stillbirths attributable to FGR decreased from 38% to 14%, in favour of other related conditions. Nearly half of SGA stillbirths (49%) were reclassified. There was a non-significant tendency toward moderate SGA, singletons and full-term stillbirths to older mothers being reclassified.ConclusionsThe position of FGR in hierarchical stillbirth classification has a major impact on the first condition associated with stillbirth. RECODE-R calls less attention to monitoring SGA fetuses but illustrates the diversity of death-related conditions for small fetuses.

A Model of the Structural and Functional Development of the Normal Human Fetal Left Ventricle Based on a Global Growth Law

The purpose of this research is to study the growth of the normal human left ventricle (LV) during the fetal period from 14 to 40 weeks of gestation. A new constitutive law for the active myocardium describing the mechanical properties of the active muscle during the whole cardiac cycle has been proposed. The LV model is a thick-walled, incompressible, hyperelastic cylinder, with families of helicoidal fibers running on cylindrical surfaces [1] . Based on the works of Lin and Taber [2] done on the embryonic chick heart, we use for the human fetal heart a growth law in which the growth rate depends on the wall stresses. The parameters of the growth law are adapted to agree with sizes and volumes inferred from two dimensional ultrasound measurements performed on 18 human fetuses. Then calculations are performed to extrapolate the cardiac performance during normal growth of the fetal LV. The results presented support the idea that a growth law in which the growth rate depends linearly on the mean wall stresses averaged through the space and during whole cardiac cycle, is adapted to the normal human fetal LV development.

Very High-Resolution Imaging of Post-Mortem Human Cardiac Tissue Using X-Ray Phase Contrast Tomography

This paper investigates the 3D microscopic structure of ex-vivo human cardiac muscle. Usual 3D imaging techniques such as DMRI or CT do not achieve the required resolution to visualise cardio-myocytes, therefore we employ X-ray phase contrast micro-CT, developed at the European Synchrotron Radiation Facility (ESRF). Nine tissue samples from the left ventricle and septum were prepared and imaged at an isotropic resolution of 3.5 (upmu )m, which is sufficient to visualise cardio-myocytes. The obtained volumes are compared with 2D histological examinations, which serve as a basis for interpreting the 3D X-ray phase-contrast results. Our experiments show that 3D X-ray phase-contrast micro-CT is a viable technique for investigating the 3D arrangement of myocytes ex-vivo at a microscopic level, allowing a better understanding of the 3D cardiac tissue architecture.

190‐kb duplication in 1p36.11 including PIGV and ARID1A genes in a girl with intellectual disability and hexadactyly

To the Editor : Coffin–Siris syndrome (CSS) is a rare autosomal dominant syndrome characterized by intellectual disability, growth deficiency, microcephaly, coarse facial features and hypoplastic nail of the fifth finger and/or toe (1). Germline mutations in ARID1A, one of the SWItch/Sucrose NonFermenting complex subunit genes, were recently identified in about 90% of CSS patients (2). Hyperphosphatasia mental retardation syndrome (HPMR), also known as Mabry syndrome, is an autosomal recessive syndrome which was first related to the triad: developmental disability, seizures and hyperphosphatasia manifesting in the first year of life (3). Within this broad phenotype, Thompson et al. delineated a specific clinical entity characterized by facial gestalt including long palpebral fissures, a broad nasal bridge and tip, a tented mouth, and brachytelephalangy (4). Homozygous and compound heterozygous missense mutations in PIGV , the first gene associated with Mabry syndrome and encoding the second mannosyltransferase in the GPI-anchor biosynthesis pathway, were identified using wholeexome sequencing in HPMR families (5). Here, we report for the first time, the clinical and molecular characterization of a patient with de novo 1p36.11 microduplication including PIGV and ARID1A. The propositus is a girl born at 41 weeks after a normal delivery from non-consanguineous healthy parents with no family history of congenital anomalies or developmental delay. Prenatal ultrasonography performed in the 22nd week of gestation had revealed a four-limb postaxial hexadactyly. The supernumerary digits were removed by surgery at 4 months (Fig. 1a,b). Her birth weight was 3180 g (> −1 SD), length 46 cm (−2 SD), and head circumference 33 cm (−1 SD). To date (3 years), her height is 87 cm (−2 SD), weight 11 kg (−2 SD), and head circumference 44 cm (< −2 SD). At 9 months old, she presented repetitive and stereotyped upper limbs movements (see Video S1, Supporting information). She had facial dysmorphic features including broad nasal bridge and tip, short philtrum, thin upper lip, abnormal ears, spare scalp hairs (Fig. 1c,d) associated with severe microcephaly of prenatal onset and overlapping toes (Fig. 1b). She also suffered of constipation, gastro-oesophageal reflux, feeding problems and eczema in relation to a cow’s milk protein allergy. She had motor skills delay with no sign of walking or crawling at 36 months. The sitting position was acquired at the age of 12 months. She had severe developmental and speech delay with only two words at 2 years old. Complete clinical and radiological examinations showed no specific abnormalities at the age of 3. Serum alkaline phosphatase level was normal (180 IU/l). No lysosomal storage and intracellular inclusions in cultured fibroblasts were observed after periodic acid Schiff reaction like those reported in the Mabry syndrome (6). It is notable that a duplication in PIGV – one of genes inactivated in Mabry syndrome – does not result in alterations in alkaline phosphatase activity in 1p36.11 duplication syndrome. Array comparative genomic hybridization analyses (CGH Microarray Kit 180K, Agilent, CA) showed a 190-kb duplication in 1p36.11 extending from base 27,001,256 to 27,190,935 (NCBI, hg 19) from the 1p telomere (Fig. 1e). No other abnormalities larger than three probes were observed, excluding well-known benign copy number variation reported in the Database of Genomic Variants (DGV). Custom multiplex ligation-dependent probe amplification (MLPA) analysis confirmed the duplication in the patient (Fig. 1f) and analysis of the parents revealed normal MLPA profiles. The fluorescent in situ hybridization analysis showed the tandem duplication in the 1p36.11 region (Fig. 1g). Reverse transcriptase multiplex ligation-dependent probe amplification analysis of mRNAs in fibroblast cell revealed that expression of ARID1A and PIGV was respectively about 1.5and 3-fold higher compared to controls (Fig. 1h). In this de novo 190-kb duplicated region, four known protein-coding genes are listed in NCBI build 37.2. Among them, ARID1A, PIGV , ZDHHC18 , SFN are completely duplicated. ZDHHC18 , whose function is unknown, is expressed predominantly in lymphoid tissue. SFN encodes for the stratifin involved in multiple cellular processes and commonly silenced in various cancers (7). In DGV, variation 4218 described many copy number variations including ZDHHC18 and SFN , suggesting that ZDHHC18 and SFN duplications are likely benign, although we cannot formally exclude their contribution in the phenotype.