TTC5 syndrome: Clinical and molecular spectrum of a severe and recognizable condition.

Biallelic mutations in the TTC5 gene have been associated with autosomal recessive intellectual disability (ARID) and subsequently with an ID syndrome including severe speech impairment, cerebral atrophy, and hypotonia as clinical cornerstones. A TTC5 role in IDs has been proposed based on the physical interaction of TTC5 with p300, and possibly reducing p300 co-activator complex activity, similarly to what was observed in Menke-Hennekam 1 and 2 patients (MKHK1 and 2) carrying, respectively, mutations in exon 30 and 31 of CREBBP and EP300, which code for the TTC5-binding region. Recently, TTC5-related brain malformation has been linked to tubulinopathies due to the function of TTC5 in tubulins' dynamics. We reported seven new patients with novel or recurrent TTC5 variants. The deep characterization of the molecular and phenotypic spectrum confirmed TTC5-related disorder as a recognizable, very severe neurodevelopmental syndrome. In addition, other relevant clinical aspects, including a severe pre- and postnatal growth retardation, cryptorchidism, and epilepsy, have emerged from the reversal phenotype approach and the review of already published TTC5 cases. Microcephaly and facial dysmorphism resulted in being less variable than that documented before. The TTC5 clinical features have been compared with MKHK1 published cases in the hypothesis that clinical overlap in some characteristics of the two conditions was related to the common p300 molecular pathway.

Biallelic Mutations in Ubiquitin-Specific Peptidase 53 (USP53) Causing Progressive Intrahepatic Cholestasis. Report of a Case With Review of Literature.

Whole-exome sequencing studies have recently identified novel genes implicated in normal- or low-GGT pediatric cholestasis including ubiquitin-specific peptidase 53 (USP53). We identified novel biallelic mutations in the USP53 gene in a 7-month-old infant with pruritus and progressive intrahepatic cholestasis. His liver biopsy showed portal and perivenular fibrosis with bland bilirubinostasis. His parents were asymptomatic heterozygous for the same mutation. He is currently on vitamin supplements and cholestyramine and his family has also been counseled for liver transplantation. Our report confirms that patients with biallelic mutation in USP53 develop cholestatic liver disease.

Biallelic mutations of CFAP58 are associated with multiple morphological abnormalities of the sperm flagella.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe type of teratozoospermia. In this study, whole-exome sequencing was conducted on 55 patients with MMAF, and biallelic mutations of CFAP58 were identified in two patients. The variants are rare and pathogenic, and CFAP58 was absent in the CFAP58-mutated sperm. The F037/II:1 couple benefited from intracytoplasmic sperm injection (ICSI). This study further indicated that CFAP58 is a pathogenic gene associated with MMAF and ICSI is an effective treatment.

Biallelic mutations in KATNAL2 cause male infertility due to oligo-astheno-teratozoospermia.

Oligo-astheno-teratozoospermia (OAT) is a common cause of male infertility, and most of idiopathic OAT patients are thought to be caused by genetic defects. Here, we recruited 38 primary infertile patients with the OAT phenotype and 40 adult men with proven fertility for genetic analysis and identified biallelic mutations of KATNAL2 by whole-exome sequencing in two cases. F013/II:1, from a consanguineous family, carried the KATNAL2 c.328C > T:p.Arg110X homozygous mutations. The other carried c.55A > G: p.Lys19Glu and c.169C > T: p Arg57Trp biallelic mutations. None of the KATNAL2 variants were found in the 40 adult men with proven fertility. The spermatozoa from patients with KATNAL2 biallelic mutations exhibited conspicuous defects in maturation, head morphology, and the structure of mitochondrial sheaths and flagella. KATNAL2 was mainly expressed in the pericentriolar material and mitochondrial sheath of the spermatozoa from control subjects, but it was undetectable in the spermatozoa from the patients. Furthermore, Katnal2 null male mice were infertile and displayed an OAT phenotype. Our results proved that the biallelic mutations in KATNAL2 cause male infertility and OAT in humans for the first time, to our knowledge, which could enrich the genetic defect spectrum of OAT and be beneficial for its accurate genetic screening and clinical diagnosis.

DNAH17 is associated with asthenozoospermia and multiple morphological abnormalities of sperm flagella.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is one kind of severe asthenozoospermia, which is caused by dysplastic development of sperm flagella. In our study, we sought to investigate the novel gene mutations leading to severe asthenozoospermia and MMAF. METHODS AND MATERIALS: The patient's spermatozoa were tested by Papanicolaou staining and transmission electron microscopy. Whole exome sequencing was performed on the patient with severe asthenozoospermia and MMAF. Sanger sequencing verified the mutations in the family. The expression of DNAH17 was detected by immunofluorescence and Western blot. RESULTS: Spermatozoa sample from the patient showed severe asthenozoospermia and MMAF. We detected biallelic mutations (c.C4445T, p.A1482V and c.C6857T, and p.S2286L) in DNAH17 (MIM:610063). The protein expression of DNAH17 was almost undetectable in spermatozoa from the patient with the biallelic mutations. CONCLUSION: These results demonstrated that DNAH17 may be involved in severe asthenozoospermia and MMAF.

Biallelic mutations in Sperm flagellum 2 cause human multiple morphological abnormalities of the sperm flagella (MMAF) phenotype.

Male patients with multiple morphological abnormalities of the sperm flagella (MMAF) are infertile and exhibit absent, short, coiled, bent and/or irregular sperm flagella. Mutations in the SPEF2 gene reduce sperm motility and cause sperm tail defects in animal models and humans. In the present study, we performed a genetic analysis on an MMAF patient and identified novel biallelic mutations in the SPEF2 gene. The biallelic mutations were confirmed by Sanger sequencing and in silico analysis revealed that, these variations were deleterious. The expression of truncated SPEF2 protein was reduced significantly in the patient's spermatozoa. The spermatozoa harbored biallelic mutations and showed severe ultrastructural defects in the axoneme and mitochondrial sheath. Our data suggest that biallelic mutations in SPEF2 can cause severe sperm flagellum defects, thus providing a novel candidate genetic pathogen for the human MMAF phenotype.

Bialleleic PKD1 mutations underlie early-onset autosomal dominant polycystic kidney disease in Saudi Arabian families.

BACKGROUND: Polycystic kidney disease (PKD) is one of the most common genetic renal diseases and may be inherited in an autosomal dominant or autosomal recessive pattern. Pathogenic variants in two major genes, PKD1 and PKD2, and two rarer genes, GANAB and DNAJB11, cause autosomal dominant PKD (ADPKD). Early onset and severe PKD can occur with PKD1 and PKD2 pathogenic variants and such phenotypes may be modified by second alleles inherited in trans. Homozygous or compound heterozygous hypomorphic PKD1 variants may also cause a moderate to severe disease PKD phenotype. METHODS: Targeted renal gene panel followed by Sanger sequencing of PKD1 gene were employed to investigate molecular causes in early onset PKD patients. RESULTS: In this study, we report four consanguineous Saudi Arabian families with early onset PKD which were associated with biallelic variants in PKD1 gene. CONCLUSIONS: Our findings confirm that PKD1 alleles may combine to produce severe paediatric onset PKD mimicking the more severe autosomal recessive ciliopathy syndromes associated with PKD. Screening of parents of such children may also reveal subclinical PKD phenotypes.

Novel biallelic SZT2 mutations in 3 cases of early-onset epileptic encephalopathy.

The seizure threshold 2 (SZT2) gene encodes a large, highly conserved protein that is associated with epileptogenesis. In mice, Szt2 is abundantly expressed in the central nervous system. Recently, biallelic SZT2 mutations were found in 7 patients (from 5 families) presenting with epileptic encephalopathy with dysmorphic features and/or non-syndromic intellectual disabilities. In this study, we identified by whole-exome sequencing compound heterozygous SZT2 mutations in 3 patients with early-onset epileptic encephalopathies. Six novel SZT2 mutations were found, including 3 truncating, 1 splice site and 2 missense mutations. The splice-site mutation resulted in skipping of exon 20 and was associated with a premature stop codon. All individuals presented with seizures, severe developmental delay and intellectual disabilities with high variability. Brain MRIs revealed a characteristic thick and short corpus callosum or a persistent cavum septum pellucidum in each of the 2 cases. Interestingly, in the third case, born to consanguineous parents, had unexpected compound heterozygous missense mutations. She showed microcephaly despite the other case and previous ones presenting with macrocephaly, suggesting that SZT2 mutations might affect head size.

Compound heterozygous mutations in COL1A1 associated with an atypical form of type I osteogenesis imperfecta.

Heterozygous mutations in the genes encoding the proα1(I) or proα2(I) chains of type I procollagen (COL1A1 and COL1A2, respectively) account for most cases of osteogenesis imperfecta (OI), a disorder characterized by reduced bone strength and increased fracture risk. COL1A1 mutations can also cause rare cases of Ehlers-Danlos syndrome (EDS), a disorder that primarily affects connective tissue and often includes reduced bone mass. Here we present a kindred of three young siblings ages 1-4 years old whose mother has a history of mild type I OI. All three children are compound heterozygotes for COL1A1 mutations, with a novel frameshift mutation (c.2522delC; p.Pro841Leufs*266) from their mother and a known missense mutation (c.3196C>T; p.R1066C) from their clinically unaffected father, which has previously been described as causing a combined type I OI/EDS phenotype. The three children exhibit features of both COL1A1 mutations: early and frequent long bone fractures, joint hyperextensibility, and blue sclerae. We describe three siblings who are the first reported surviving subjects with biallelic pathogenic COL1A1 mutations. They have a more severe form of type I OI with features of EDS that represents their compound heterozygosity for two deleterious COL1A1 mutations. Their long-term outcomes are yet to be determined.

Biallelic mutations in huntington disease: A new case with just one affected parent, review of the literature and terminology.

Patients with biallelic mutations for Huntington disease (HD) are rare. We present a 46-year-old female with two expanded Huntingtin (HTT) alleles with just one known affected parent. This is the first reported patient with molecular studies performed to exclude HTT uniparental disomy (UPD). The proband had biparental inheritance of HTT alleles (42/44 CAG repeats). Given the negative UPD results, the proband's unaffected mother either had a reduced penetrance allele that expanded into the full mutation range during transmission to our patient or an unknown full HTT mutation and died before symptom onset, unlikely given no family history of HD and asymptomatic at age 59. We made the novel observation in our literature review that most patients with biallelic HD did not have two full HTT mutations. Most had one HTT allele that was in the intermediate or reduced penetrance ranges or 40 CAG repeats, the lowest limit of the full mutation range. Although the number of patients is small, when an allele in these size ranges was present, generally the age of HD onset was in the 50s. If the second HTT allele had >45 repeats, then onset was typically 20s-30s. While similar ages of onset have been reported for patients with one or two HTT mutations, patients with biallelic mutations may have later onset if an expanded HTT allele has ≤40 CAG repeats. Finally, we propose that "biallelic mutations" or "compound heterozygosity" are more accurate descriptive terms than "homozygosity" when there are two non-identical expanded HTT alleles.

Recessive GNE Mutations in Korean Nonaka Distal Myopathy Patients with or without Peripheral Neuropathy.

Autosomal recessive Nonaka distal myopathy is a rare autosomal recessive genetic disease characterized by progressive degeneration of the distal muscles, causing muscle weakness and decreased grip strength. It is primarily associated with mutations in the GNE gene, which encodes a key enzyme of sialic acid biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase). This study was performed to find GNE mutations in six independent distal myopathy patients with or without peripheral neuropathy using whole-exome sequencing (WES). In silico pathogenic prediction and simulation of 3D structural changes were performed for the mutant GNE proteins. As a result, we identified five pathogenic or likely pathogenic missense variants: c.86T>C (p.Met29Thr), c.527A>T (p.Asp176Val), c.782T>C (p.Met261Thr), c.1714G>C (p.Val572Leu), and c.1771G>A (p.Ala591Thr). Five affected individuals showed compound heterozygous mutations, while only one patient revealed a homozygous mutation. Two patients revealed unreported combinations of combined heterozygous mutations. We observed some specific clinical features, such as complex phenotypes of distal myopathy with distal hereditary peripheral neuropathy, an earlier onset of weakness in legs than that of hands, and clinical heterogeneity between two patients with the same set of compound heterozygous mutations. Our findings on these genetic causes expand the clinical spectrum associated with the GNE mutations and can help prepare therapeutic strategies.

Identification of biallelic mutations in MCM3AP and comprehensive literature analysis.

Background: Minichromosome maintenance complex component 3 associated protein (MCM3AP) is a gene in which mutations can result in autosomal recessive peripheral neuropathy with or without impaired intellectual development. The MCM3AP genotype-phenotype correlation and prognosis remain unclear. The aim of this study was to explore the genotype-phenotype correlations pertaining to MCM3AP. Methods: Whole-exome sequencing (WES) combined with copy number variation sequencing (CNV-seq) were performed on the genomic DNA isolated from a Chinese family, and Sanger sequencing, quantitative PCR and cDNA analyses were performed to examine the mutations. The retrospective study was conducted on 28 individuals with biallelic MCM3AP mutation-related diseases, including features such as mutations, motor development impairment, intellectual disability, weakness/atrophy, and cerebral magnetic resonance imaging abnormalities. Results: Sequencing identified novel compound heterozygous mutations in MCM3AP, namely, a paternal variant c.1_5426del (loss of exons 1-25) and a maternal splicing variant c.1858 + 3A>G. Functional studies revealed that the variant c.1858 + 3A>G resulted in the heterozygous deletion of exon 5, thereby affecting splicing functionality. Furthermore, the compound heterozygous mutation may affect the functionality of the protein domain. Retrospective analysis revealed different genotype-phenotype correlations for the pathogenic variants in biallelic MCM3AP: all individuals (100%) with mutations outside the Sac3 domain exhibited early-onset symptoms, motor developmental delays, and cognitive abnormalities, conversely, the proportions of individuals carrying mutations within the domain were 26.7% (motor delays) and 46.7% (cognitive abnormalities). Conclusion: Our findings further expand the genetic mutation spectrum of biallelic MCM3AP and highlight the genotype-phenotype associations. Additionally, we elaborate on the importance of rehabilitation intervention.

A family with nine siblings showing signs of Rothmund-Thomson syndrome with two being definitely diagnosed with the syndrome due to homozygous N-terminal mutation of RECQL4.

This study presents a family with nine children, two of them diagnosed with RTS2 using genetic testing. The other siblings show some of the RTS2 criteria and are suggestive of the syndrome. Such reports help physicians be more alert in dealing with cases of rare syndromes. Timely initiation of genetic counseling and testing once the first child was diagnosed with the syndrome could have prevented the birth of affected siblings by RTS2. Since RTS2 patients could have a severe clinical manifestation as osteosarcoma which probably leads to death at a young age, the importance of genetic testing is even more underlined.

Case report: biallelic DNMT3A mutations in acute myeloid leukemia.

DNMT3A gene mutations, detected in 20-25% of de novo acute myeloid leukemia (AML) patients, are typically heterozygous. Biallelic variants are uncommon, affecting ~3% of cases and identifying a worse prognosis. Indeed, two concomitant DNMT3A mutations were recently associated with shorter event-free survival and overall survival in AML. We present an AML case bearing an unusual DNMT3A molecular status, strongly affecting its function and strangely impacting the global genomic methylation profile. A 56-year-old Caucasian male with a diagnosis of AML not otherwise specified (NOS) presented a complex DNMT3A molecular profile consisting of four different somatic variants mapping on different alleles (in trans). 3D modelling analysis predicted the effect of the DNMT3A mutational status, showing that all the investigated mutations decreased or abolished DNMT3A activity. Although unexpected, DNMT3A's severe loss of function resulted in a global genomic hypermethylation in genes generally involved in cell differentiation. The mechanisms through which DNMT3A contributes to AML remain elusive. We present a unique AML case bearing multiple biallelic DNMT3A variants abolishing its activity and resulting in an unexpected global hypermethylation. The unusual DNMT3A behavior described requires a reflection on its role in AML development and persistence, highlighting the heterogeneity of its deregulation.

Diagnosing Alström syndrome in a patient followed up with syndromic obesity for years.

Alström syndrome (AS) is a rare autosomal recessive monogenic disorder caused by mutations of the Alström syndrome 1 (ALMS1) gene, located on chromosome 2p13. It is a progressive multisystemic disease characterized mostly by obesity, sensorineural hearing loss, visual impairments, cardiomyopathy, insulin resistance and/or type 2 diabetes mellitus (T2DM), metabolic dysfunctions, non-alcoholic fatty liver disease, and chronic progressive kidney disease. Generally, the first clinical symptoms of the disease appear in the first years of life with a major variation of onset age. In this study, we aimed to examine the molecular diagnosis of a 6-year-old patient with suspected AS clinical symptoms. After applying clinical exome sequencing (CES) in the patient we found a homozygous deletion in exon 8 at the ALMS1 gene (c.2311_2312del). We identified a homozygous frameshift mutation. The reported variant was pathogenic according to the criteria of the American College of Medical Genetics and Genomics (ACMG). Thus, the patient was diagnosed with AS as a result of the combined clinical phenotype and genetic tests results. We hope the variant we found can expand the spectrum of ALMS1 variants in AS.

Two novel biallelic mutations in PSMC3IP in a patient affected by premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a heterogeneous condition occurring when a woman experiences a loss of ovarian activity before the age of 40. POI is one of the most common reproductive endocrine diseases in women of childbearing age. The present study investigated the clinical manifestations and genetic features of a Chinese patient affected by POI. Next­generation whole­exome capture sequencing with Sanger direct sequencing were applied to the proband and her clinically unaffected family members. Two novel compound heterozygous mutations were identified in PSMC3IP. The first was a splicing mutation (c.597+1G>T) that was inherited from her father, whereas the second mutation (c.268G>C p.D90H) was discovered in both her mother and younger sister. The two mutations were co­segregated with the disease phenotype in the family. In conclusion, the findings of the present study further support the key role of PSMC3IP in the etiology of POI and provide a novel insight into elucidating the mechanisms of female infertility.

Biallelic mutations in spermatogenesis and centriole-associated 1 like (SPATC1L) cause acephalic spermatozoa syndrome and male infertility.

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

Biallelic mutations in DCDC2 cause neonatal sclerosing cholangitis in a Chinese family.

BACKGROUND: Neonatal sclerosing cholangitis (NSC) is a severe cholestatic liver disease, which often develops into end-stage liver disease in childhood and requires liver transplantation. Mutations in CLDN1 and DCDC2 are confirmed to be the main pathogenic mechanism of NSC. METHODS: Whole exon sequencing (WES) was performed to find the possible disease-causing mutations of this family. The mutation was confirmed by Sanger sequencing, and large fragment copy number variation was confirmed by qPCR. RESULTS: We found novel biallelic mutations c.[705-2A>G];[923_1023del] in the DCDC2 gene of the proband. The proband's father had the heterozygous mutation c.705-2A>G, and his mother had a heterozygous c.923_1023del. The proband's younger brother, who had similar clinical manifestations, was found the same biallelic mutations with the proband. CONCLUSION: Novel biallelic mutations were identified in DCDC2 of this Chinese family, according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants, both mutations were classified as pathogenic, which might be the cause of NSC in this family.

Biallelic TOR1A mutations cause severe arthrogryposis: A case requiring reverse phenotyping.

Heterozygous mutations in TOR1A gene are known to be responsible for DYT1 dystonia with incomplete penetrance. Autosomal recessive TOR1A disease is a very recently described syndrome characterized by severe arthrogryposis, developmental delay, strabismus and tremor. A 2 month-old boy with severe arthrogryposis and developmental delay was referred to our department for genetic counseling. Dystonic movements were observed on physical examination. Whole exome sequencing revealed a homozygous nonsense variant in exon 5 of TOR1A (c.862C > T, p.Arg288*). Our results expand the phenotypic and mutational spectrum of biallelic TOR1A disease, while emphasizing the importance of reverse phenotyping in the diagnosis of rare genetic disorders.