Leukoencephalopathy and early death associated with an Ashkenazi-Jewish founder mutation in the Hikeshi gene.

BACKGROUND: Leukodystrophies are genetic white matter disorders affecting the formation or maintenance of myelin. Among the recently discovered genetic defects associated with leukodystrophies, several genes converge on a common mechanism involving protein transcription/translation and ER stress response. METHODS: The genetic basis of a novel congenital leukodystrophy, associated with early onset spastic paraparesis, acquired microcephaly and optic atrophy was studied in six patients from three unrelated Ashkenazi-Jewish families. To this end we used homozygosity mapping, exome analysis, western blot (Hikeshi, HSF1-pS326 and b-actin) in patient fibroblasts, indirect immunofluorescence (HSP70 and HSF1) in patient fibroblasts undergoing heat shock stress, nuclear injection of plasmids expressing Hikeshi or EGFP in patient fibroblasts, in situ hybridization and Immunoblot analysis of Hikeshi in newborn and adult mouse brain. RESULTS: All the patients were homozygous for a missense mutation, p.Val54Leu, in C11ORF73 encoding HSP70 nuclear transporter protein, Hikeshi. The mutation segregated with the disease in the families and was carried by 1:200 Ashkenazi-Jewish individuals. The mutation was associated with undetectable level of Hikeshi in the patients' fibroblasts and with lack of nuclear HSP70 during heat shock stress, a phenomenon which was reversed upon the introduction of normal human Hikeshi to the patients cells. Hikeshi was found to be expressed in central white matter of mouse brain. CONCLUSIONS: These data underscore the importance of Hikeshi for HSP70 relocation into the nucleus. It is likely that in the absence of Hikeshi, HSP70 cannot attenuate the multiple heat shock induced nuclear phenotypes, leaving the cells unprotected during heat shock stress. We speculate that the sudden death of three of the six patients following a short febrile illness and the life-threatening myo-pericarditis in the fourth are the result of excess extra-nuclear HSP70 level which initiates cytokine release or provide target for natural killer cells. Alternatively, nuclear HSP70 might play an active role in stressed cells protection.

Lack of association of KATNAL1 gene sequence variants and azoospermia in humans.

PURPOSE: A recent experiment indicated that a loss of function mutation in the murine Katnal1 gene resulted in male factor infertility due to premature exfoliation of spermatids. This study investigated the relevance of this gene to infertility in humans. METHODS: Multiple methods of genetic analysis were employed to investigate whether mutations in human KATNAL1 have a causative role in male infertility. This was a genetic association study, which included DNA samples from 105 men with non-obstructive azoospermia (NOA) and 242 anonymous sperm donor controls. 28 commercially available TaqMan SNP assays were used to haplotype samples from both groups and genetically tag regions of interest across the entire gene. AmpliSeq primers were then designed for identified regions so that targeted next-generation sequencing (NGS) could be used to identify causative variants. RESULTS: Four SNPs in the 3'UTR demonstrated a putative association with NOA. The AmpliSeq primers designed for the 3'UTR provided 83 % coverage of the 7,202 basepairs within the regions of interest. Variant sites were analyzed against genetic models to identify sequence polymorphisms which associated with NOA. No variants met standard criteria for significance when tested between the groups. CONCLUSIONS: This study suggests a lack of association of KATNAL1 gene sequence variants and azoospermia in humans.

Prenatal Diagnosis of Cystic Fibrosis.

Cystic fibrosis (CF) is an inherited disease characterized by the accumulation of thick, sticky mucus which damages epithelia in organs such as the lungs, pancreas, liver, intestines, and other parts of the body. The most common symptoms are sinopulmonary disease and chronic gastrointestinal tract problems resulting from decreased mucociliary clearance and inflammation. CF is the most common life-limiting autosomal recessive disorder in people of northern European ancestry and it affects other populations with different prevalence. CF can be diagnosed by many methods including testing for blood immunoreactive trypsin, sweat chloride, transepithelial nasal potential difference, and molecular genetic testing.

Identification of a novel pathogenic OTOF variant causative of nonsyndromic hearing loss with high frequency in the Ashkenazi Jewish population.

Mutations in the OTOF gene have previously been shown to cause nonsyndromic prelingual deafness (DFNB9, OMIM 601071) as well as auditory neuropathy/dys-synchrony. In this study, the OTOF NM_194248.2 c.5332G>T, p.Val1778Phe variant was identified in a large Ashkenazi Jewish family as the causative variant in four siblings with hearing loss. Our analysis reveals a carrier frequency of the OTOF c.5332G>T, p.Val1778Phe variant of 1.27% in the Ashkenazi Jewish population, suggesting that this variant may be a significant contributor to nonsyndromic sensorineural hearing loss and should be considered for inclusion in targeted hearing loss panels for this population. Of note, the degree of hearing loss associated with this phenotype ranged from mild to moderately severe, with two of the four siblings not known to have hearing loss until they were genotyped and underwent pure tone audiometry and auditory brainstem response testing. The phenotypic variability along with the auditory neuropathy/dys-synchrony, which allows for the production of otoacoustic emissions, supports that nonsyndromic hearing loss caused by OTOF mutations may be much more common in the Ashkenazi Jewish population than currently appreciated due to a lack of diagnosis.

Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.

OBJECTIVE: To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). DESIGN: Prospective and blinded. SETTING: Not applicable. PATIENT(S): Couples indicated for preimplantation genetic diagnosis (PGD). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. RESULT(S): The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33). CONCLUSION(S): This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification.

Carrier frequencies of eleven mutations in eight genes associated with primary ciliary dyskinesia in the Ashkenazi Jewish population.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder that results from functional and ultrastructural abnormalities of motile cilia. Patients with PCD have diverse clinical phenotypes that include chronic upper and lower respiratory tract infections, situs inversus, heterotaxy with or without congenital heart disease, and male infertility, among others. In this report, the carrier frequencies for eleven mutations in eight PCD-associated genes (DNAI1, DNAI2, DNAH5, DNAH11, CCDC114, CCDC40, CCDC65, and C21orf59) that had been found in individuals of Ashkenazi Jewish descent were investigated in order to advise on including them in existing clinical mutation panels for this population. Results showed relatively high carrier frequencies for the DNAH5 c.7502G>C mutation (0.58%), the DNAI2 c.1304G>A mutation (0.50%), and the C21orf59 c.735C>G mutation (0.48%), as well as lower frequencies for mutations in DNAI1, CCDC65, CCDC114, and DNAH11 (0.10-0.29%). These results suggest that several of these genes should be considered for inclusion in carrier screening panels in the Ashkenazi Jewish population.