Exome sequencing reveals HINT1 mutations as a cause of distal hereditary motor neuropathy

Distal hereditary motor neuropathies (dHMNs) are a heterogenous group of genetic disorders with length-dependent degeneration of motor axons. Obtaining a genetic diagnosis in patients with dHMN remains challenging. We performed exome sequencing in a diagnostic setting in 12 patients with a clinical diagnosis of dHMN. Potential disease-causing variants in genes associated with dHMN and other forms of inherited neuropathies/motor neuron diseases were validated using Sequenom. The coverage in the genes studied was >95% with an average coverage of >50 times. In none of the patients a mutations was found in genes previously reported to be associated with dHMN. However, in 2/12 patients a recessive mutation in histidine triad nucleotide binding protein 1 (HINT1, recently discovered as a cause of axonal neuropathy with neuromyotonia) was identified. Our results demonstrate the diagnostic value of exome sequencing for patients with inherited neuropathies. The phenotypic spectrum of recessive mutations in HINT1 includes dHMN. HINT1 should be added to the list of genes to check for in dHMN.     

A novel PTC mutation in the BTB domain of KLHL7 gene in two patients with Bohring-Opitz syndrome-like features

The bric-a-brac, tramtrack and broad complex (BTB) superfamily of conserved proteins are involved in ubiquitin-proteasome system that contains the Kelch-like (KLHL) gene family. Kelch-like family member 7 (KLHL7), one of the KLHL gene family, consists of one BTB/POZ domain, one BACK domain and five or six Kelch motifs. Numerous variants in KLHL7 gene domains have been reported with Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1)-like features and retinitis pigmentosa 42, and have recently been identified as causing Bohring-Opitz syndrome (BOS)-like features. We report two siblings with BOS-like phenotype with healthy parents and living in Qazvin province (Central Iran).We performed whole-exome sequencing (WES) on the older patient and Sanger sequencing was carried out for validation of potential causative variants in the close family.A novel homozygous frameshift mutation, p.(Phe83Leufs*3), was identified in the BTB domain of KLHL7 that caused a premature translation-termination codon (PTC) in the two siblings with severe developmental delay, microcephaly, facial dysmorphism, peripheral retinal and optic disc atrophy and cardiac septal defects.Our findings are in agreement with the clinical spectrum of KLHL7 mutations, which are associated with BOS-like features that reports for first time in our population.     

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

Congenital disorders of glycosylation (CDG) are a large group of recessive multisystem disorders caused by impaired protein or lipid glycosylation. The CDG-I subgroup is characterized by protein N-glycosylation defects originating in the endoplasmic reticulum. The genetic defect is known for 17 different CDG-I subtypes. Patients in the few reported DPAGT1-CDG families exhibit severe intellectual disability (ID), epilepsy, microcephaly, severe hypotonia, facial dysmorphism and structural brain anomalies. In this study, we report a non-consanguineous family with two affected adults presenting with a relatively mild phenotype consisting of moderate ID, epilepsy, hypotonia, aggressive behavior and balance problems. Exome sequencing revealed a compound heterozygous missense mutation, c.85A>T (p.I29F) and c.503T>C (p.L168P), in the DPAGT1 gene. The affected amino acids are located in the first and fifth transmembrane domains of the protein. Isoelectric focusing and high-resolution mass spectrometry analyses of serum transferrin revealed glycosylation profiles that are consistent with a CDG-I defect. Our results show that the clinical spectrum of DPAGT1-CDG is much broader than appreciated so far.     

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox–Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor.     

Whole-exome sequencing identifies rare compound heterozygous mutations in the MSTO1 gene associated with cerebellar ataxia and myopathy

Human MSTO1 is involved in the regulation of mitochondrial distribution and morphology and its unregulated expression leads to mitochondrial disorder. Despite its significance for mitochondrial functions, human MSTO1 gene is rarely studied before 2017. As of late, MSTO1 mutations have been reported to cause clinical manifestations such as myopathy, cerebellar atrophy and ataxia, motor developmental delay, and pigmentary retinopathy. Here we have performed a whole-exome sequencing in a family which includes two brothers showing cerebellar atrophy and ataxia, intellectual disability, and myopathy. As a result, two mutations were identified. One of these mutations has been identified as a missense mutation, c.836G > A; p. (Arg279His) and a novel frameshift variant, c.1259delG; p. (Gly420ValfsTer2). So, the two brothers both had compound heterozygous mutations with a combination of protein-truncation mutation and missense mutation. These findings suggested an association of MSTO1 mutations with the early onset of symptoms and revealed the genotype-phenotype correlation between different mutation cases. In this case, the two brothers both have pes planus which is not reported in other cases. This might suggest that the novel mutation is responsible for dysmorphia. Thus, the recessive and novel MSTO1 mutations enriches genetic information on the pathogenicity of MSTO1 in humans.     

Exome sequencing identified a de novo mutation of PURA gene in a patient with familial Xp22.31 microduplication

The clinical significance of Xp22.31 microduplication is controversial as it is reported in subjects with developmental delay (DD), their unaffected relatives and unrelated controls. We performed multifaceted studies in a family of a boy with hypotonia, dysmorphic features and DD who carried a 600?Kb Xp22.31 microduplication (7515787-8123310bp, hg19) containing two genes, VCX and PNPLA4. The duplication was transmitted from his cognitively normal maternal grandfather.We found no evidence of the duplication causing the proband's DD and congenital anomalies based on unaltered expression of PNPLA4 in the proband and his mother in comparison to controls and preferential activation of the paternal chromosome X with Xp22.31 duplication in proband's mother. However, a de novo, previously reported deleterious, missense mutation in Pur-alpha gene (PURA) (5q31.2), with a role in neuronal differentiation was detected in the proband by exome sequencing.We propose that the variability in the phenotype in carriers of Xp22.31 microduplication can be due to a second and more deleterious genetic mutation in more severely affected carriers. Widespread use of whole genome next generation sequencing in families with Xp22.31 CNV could help identify such cases.     

Diagnostic exome sequencing identifies a heterozygous MBD5 frameshift mutation in a family with intellectual disability and epilepsy

Methyl-CpG-binding domain 5 (MBD5)-associated neurodevelopmental disorder caused by 2q23.1 or MBD5-specific mutation has been recently identified as a genetic disorder associated with autism spectrum disorders. Phenotypic features of 2q23.1 deletion or disruption of MBD5 gene include severe intellectual disability, seizure, significant speech impairment, sleep disturbance, and autistic-like behavioural problems. Here we report a 7-year-old girl with intellectual disability and epilepsy without previous clinical diagnosis. Diagnostic exome sequencing identified a novel frameshift mutation c.254_255delGA (p.Arg85Asnfs*6) in the MBD5 gene of the proband and her father. The proband's father with normal intelligence showed subclinical manifestations observed in subsequent investigations. Clinical manifestations, disease course, and molecular findings of the involvement of MBD5 gene in this family suggest an unusual MBD5-related neurodevelopmental disorder. Moreover, this report demonstrates the critical role of next-generation sequencing technique in characterizing such a rare disorder with variable or no clinical manifestation and providing opportunity to develop effective preventive measures such as pre-implantation genetic diagnosis.     

Biallelic variants in four genes underlying recessive osteogenesis imperfecta

Osteogenesis imperfecta (OI) is an inherited heterogeneous rare skeletal disorder characterized by increased bone fragility and low bone mass. The disorder mostly segregates in an autosomal dominant manner. However, several rare autosomal recessive and X-linked forms, caused by mutations in 18 different genes, have also been described in the literature.Here, we present five consanguineous families segregating OI in an autosomal recessive pattern. Affected individuals in the five families presented severe forms of skeletal deformities. It included frequent bone fractures with abnormal healing, short stature, facial dysmorphism, osteopenia, joint laxity, and severe scoliosis. In order to search for the causative variants, DNA of at least one affected individual in three families (A-C) were subjected to whole exome sequencing (WES). In two other families (D-E), linkage analysis using highly polymorphic microsatellite markers was followed by Sanger sequencing. Sequence analysis revealed two novels and three previously reported disease-causing variants. The two novel homozygous variants including [c.824G > A; p.(Cys275Tyr)] in the SP7 gene and [c.397C > T, p.(Gln133*)] in the SERPINF1 gene were identified in families A and B, respectively. The three previously reported homozygous variants including [c.497G > A; p.(Arg166His)] in the SPARC gene, (c.359-3C > G; intron 2) and [c.677C > T; p.(Ser226Leu)] in the WNT1 gene were identified in family C, D, and E.In conclusion, our findings provided additional evidence of involvement of homozygous sequence variants in the SP7, SERPINF1, SPARC and WNT1 genes causing severe OI. It also highlights the importance of extensive genetic investigations to search for the culprit gene in each case of skeletal deformity.     

DNAH2 is a novel candidate gene associated with multiple morphological abnormalities of the sperm flagella

Multiple morphological abnormalities of flagella (MMAF) is one kind of severe teratozoospermia. Gene mutations reported in previous works only revealed the pathogenesis of approximately half of the MMAF cases, and more genetic defects in MMAF need to be explored. In the present study, we performed a genetic analysis on Han Chinese men with MMAF using whole-exome sequencing. After filtering out the cases with known gene mutations, we identified five novel mutation sites in the DNAH2 gene in three cases from three families. These mutations were validated through Sanger sequencing and absent in all control individuals. In silico analysis revealed that these DNAH2 variations are deleterious. The spermatozoa with DNAH2 mutations showed severely disarranged axonemal structures with mitochondrial sheath defection. The DNAH2 protein level was significantly decreased and inner dynein arms were absent in the spermatozoa of patients. ICSI treatment was performed for two MMAF patients with DNAH2 mutations and the associated couples successfully achieved pregnancy, indicating good nuclear quality of the sperm from the DNAH2 mutant patients. Together, these data suggest that the DNAH2 mutation can cause severe sperm flagella defects that damage sperm motility. These results provide a novel genetic pathogeny for the human MMAF phenotype.     

Clinical analysis of Noonan syndrome caused by RRAS2 mutations and literature review

Noonan syndrome is a common developmental disorder characterized by distinctive facial dysmorphism, short stature, congenital heart defects, pectus deformity, and developmental delay. It is related to the abnormal activation of genes involved in the RAS-MAPK signaling pathway, more than a dozen of which can be affected. However, mutations of the RRAS2 gene are rare, with only 6 different RRAS2 variants in 13 patients reported to date. In this case report, whole-exome sequencing revealed a novel heterozygous variant in the RRAS2 gene NM_012250: c.212G > A, p.(Gly71Glu). Phenotypically, our patient had typical Noonan syndrome-related clinical manifestations consistent with published reports, such as short stature, facial dysmorphism, short neck, patent foramen ovale, moderate global developmental delay, and hearing impairment. In addition, our patient also had a distal middle finger deformity and hair defect, which have not been reported in previous cases. We analyzed the clinical characteristics of all patients with Noonan syndrome caused by RRAS2 variants and reviewed the literature. This discovery expands the genetic and phenotypic spectrum of Noonan syndrome.     

Molecular genetic analysis of 30 families with Joubert syndrome

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the ‘molar tooth sign’. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole‐exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012‐12T>A (75.0%), an allele that has not been reported in non‐Japanese populations. Therefore c.6012‐12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet–Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.     

2 new cases of pontocerebellar hypoplasia type 10 identified by whole exome sequencing in a Turkish family

Pontocerebellar hypoplasia type 10 (PCH10) is a progressive autosomal recessive neurodegenerative disorder that has been recently described in association with cleavage and polyadenylation factor I subunit 1 (CLP1) mutations. To date, all reported cases have the same homozygous missense mutation in the CLP1 gene suggesting a founder mutation. CLP1 is an RNA kinase involved in tRNA splicing and maturation. There is evidence that the mutation is associated with functionally impaired kinase activity and subsequent defective tRNA processing. Through whole exome sequencing, we identified the same mutation in an extended family of Turkish origin. Both children presented with severe psychomotor delay, progressive microcephaly, and constipation. However, intrafamilial phenotypic variability is suggested due to the variability in their brain abnormalities and clinical features.     

An atypical 7q11.23 deletion in a normal IQ Williams�CBeuren syndrome patient

Williams–Beuren syndrome (WBS; OMIM no. 194050) is a multisystemic neurodevelopmental disorder caused by a hemizygous deletion of 1.55 Mb on chromosome 7q11.23 spanning 28 genes. Haploinsufficiency of the ELN gene was shown to be responsible for supravalvular aortic stenosis and generalized arteriopathy, whereas LIMK1, CLIP2, GTF2IRD1 and GTF2I genes were suggested to be linked to the specific cognitive profile and craniofacial features. These insights for genotype–phenotype correlations came from the molecular and clinical analysis of patients with atypical deletions and mice models. Here we report a patient showing mild WBS physical phenotype and normal IQ, who carries a shorter 1 Mb atypical deletion. This rearrangement does not include the GTF2IRD1 and GTF2I genes and only partially the BAZ1B gene. Our results are consistent with the hypothesis that hemizygosity of the GTF2IRD1 and GTF2I genes might be involved in the facial dysmorphisms and in the specific motor and cognitive deficits observed in WBS patients.