Charcot–Marie–Tooth disease (CMT) and autosomal recessive spastic ataxia of Charlevoix–Saguenay type (ARSACS) are large heterogeneous groups of sensory, neurological genetic disorders characterized by sensory neuropathies, muscular atrophies, abnormal sensory conduction velocities, and ataxia. CMT2EE (OMIM: 618400) is caused by mutations in MPV17 (OMIM: 137960), CMT4F (OMIM:614895) is caused by PRX (OMIM: 605725), CMTX1 (OMIM:302800) is caused by mutations in GJB1 (OMIM: 304040), and ARSACS (OMIM:270550) is caused by mutations in SACS (OMIM: 604490). In this study, we enrolled four families: DG-01, BD-06, MR-01, and ICP-RD11, with 16 affected individuals, for clinical and molecular diagnoses. One patient from each family was analyzed for whole exome sequencing and Sanger sequencing was done for the rest of the family members. Affected individuals of families BD-06 and MR-01 show complete CMT phenotypes and family ICP-RD11 shows ARSACS type. Family DG-01 shows complete phenotypes for both CMT and ARSACS types. The affected individuals have walking difficulties, ataxia, distal limb weakness, axonal sensorimotor neuropathies, delayed motor development, pes cavus, and speech articulations with minor variations. The WES analysis in an indexed patient of family DG-01 identified two novel variants: c.83G>T (p.Gly28Val) in MPV17 and c.4934G>C (p.Arg1645Pro) in SACS. In family ICP-RD11, a recurrent mutation that causes ARSACS, c.262C>T (p.Arg88Ter) in SACS, was identified. Another novel variant, c.231C>A (p.Arg77Ter) in PRX, which causes CMT4F, was identified in family BD-06. In family MR-01, a hemizygous missense variant c. 61G>C (p.Gly21Arg) in GJB1 was identified in the indexed patient. To the best of our knowledge, there are very few reports on MPV17, SACS, PRX, and GJB1 causing CMT and ARSACS phenotypes in the Pakistani population. Our study cohort suggests that whole exome sequencing can be a useful tool in diagnosing complex multigenic and phenotypically overlapping genetic disorders such as Charcot–Marie–Tooth disease (CMT) and spastic ataxia of Charlevoix–Saguenay type.

Background: Hermansky–Pudlak syndrome (HSP) was first reported in 1959 as oculocutaneous albinism with bleeding abnormalities, and now consists of 11 distinct heterogenic genetic disorders that are caused by mutations in four protein complexes: AP-3, BLOC1, BLOC2, and BLOC3. Most of the patients show albinism and a bleeding diathesis; additional features may present depending on the nature of a defective protein complex. The subtypes 3 and 4 have been known for mutations in HSP3 and HSP4 genes, respectively. Methods: In this study, two Pakhtun consanguineous families, ALB-09 and ALB-10, were enrolled for clinical and molecular diagnoses. Whole-exome sequencing (WES) of the index patient in each family followed by Sanger sequencing of all available samples was performed using 3Billion. Inc South Korea rare disease diagnostics services. Results: The affected individuals of families ALB-09 and ALB-10 showed typical phenotypes of HPS such as oculocutaneous albinism, poor vision, nystagmus, nystagmus-induced involuntary head nodding, bleeding diathesis, and enterocolitis; however, immune system weakness was not recorded. WES analyses of one index patient revealed a novel nonsense variant (NM_032383.4: HSP3; c.2766T > G) in family ALB-09 and a five bp deletion (NM_001349900.2: HSP4; c.1180_1184delGTTCC) variant in family ALB-10. Sanger sequencing confirmed homozygous segregation of the disease alleles in all affected individuals of the respective family. Conclusions: The substitution c.2766T > G creates a premature protein termination at codon 922 in HPS3, replacing tyrosine amino acid with a stop codon (p.Tyr922Ter), while the deletion mutation c.1180_1184delGTTCC leads to a reading frameshift and a premature termination codon adding 23 abnormal amino acids to HSP4 protein (p:Val394Pro395fsTer23). To the best of our knowledge, the two novel variants identified in HPS3 and HPS4 genes causing Hermansky–Pudlak syndrome are the first report from the Pakhtun Pakistani population. Our work expands the pathogenic spectrum of HPS3 and HPS4 genes, provides successful molecular diagnostics, and helps the families in genetic counselling and reducing the disease burden in their future generations.

Introduction: Epilepsy is a group of neurological disorders characterized by recurring seizures and fits. The Epilepsy genes can be classified into four distinct groups, based on involvement of these genes in different pathways leading to Epilepsy as a phenotype. Genetically the disease has been associated with various pathways, leading to pure epilepsy-related disorders caused by CNTN2 variations, or involving physical or systemic issues along with epilepsy caused by CARS2 and ARSA, or developed by genes that are putatively involved in epilepsy lead by CLCN4 variations.Methods: In this study, five families of Pakistani origin (EP-01, EP-02, EP-04, EP-09, and EP-11) were included for molecular diagnosis.Results: Clinical presentations of these patients included neurological symptoms such as delayed development, seizures, regression, myoclonic epilepsy, progressive spastic tetraparesis, vision and hearing impairment, speech problems, muscle fibrillation, tremors, and cognitive decline. Whole exome sequencing in index patients and Sanger sequencing in all available individuals in each family identified four novel homozygous variants in genes CARS2: c.655G>A p.Ala219Thr (EP-01), ARSA: c.338T>C: p.Leu113Pro (EP-02), c.938G>T p.Arg313Leu (EP-11), CNTN2: c.1699G>T p.Glu567Ter (EP-04), and one novel hemizygous variant in gene CLCN4: c.2167C>T p.Arg723Trp (EP-09).Conclusion: To the best of our knowledge these variants were novel and had not been reported in familial epilepsy. These variants were absent in 200 ethnically matched healthy control chromosomes. Three dimensional protein analyses revealed drastic changes in the normal functions of the variant proteins. Furthermore, these variants were designated as “pathogenic” as per guidelines of American College of Medical Genetics 2015. Due to overlapping phenotypes, among the patients, clinical subtyping was not possible. However, whole exome sequencing successfully pinpointed the molecular diagnosis which could be helpful for better management of these patients. Therefore, we recommend that exome sequencing be performed as a first-line molecular diagnostic test in familial cases.

BackgroundAutosomal recessive cutis laxa type 2A (ARCL2A; OMIM: 219200) is characterized by neurovegetative, developmental and progeroid elastic skin anomalies. It is caused by biallelic variation in ATPase, H+ transporting V0 subunit A2 (ATP6V0A2; OMIM: 611716) located on chromosome 12q24.31. Autosomal recessive cutis laxa type 3A (ARCL3A; OMIM: 219150) is another subclinical type characterized by short stature, ophthalmological abnormalities and a progeria‐like appearance. The ARCL3A is caused by loss of function alterations in the aldehyde dehydrogenase 18 family member A1 (ALDH18A1; OMIM: 138250) gene located at chromosome 10q24.1.MethodsWhole‐exome sequencing (WES), and Sanger sequencing were performed for molecular diagnosis. 3D protein modeling was performed to investigate the deleterious effect of the variant on protein structure.ResultsIn this study, clinical and molecular diagnosis were performed for two families, ED‐01 and DWF‐41, which displayed hallmark features of ARCL2A and ARCL3A, respectively. Three affected individuals in the ED‐01 family (IV‐4, IV‐5 and V‐3) displayed sagging loose skin, down‐slanting palpebral fissures, excessive wrinkles on the abdomen, hands and feet, and prominent veins on the trunk. Meanwhile the affected individuals in the DWF‐41 family (V‐2 and V‐3) had progeroid skin, short stature, dysmorphology, low muscle tone, epilepsy, lordosis, scoliosis, delayed puberty and internal genitalia. WES in the index patient (ED‐01: IV‐4) identified a novel homozygous deletion (NM_012463.3: c.1977_1980del; p.[Val660LeufsTer23]) in exon 16 of the ATP6V0A2 while in DWF‐41 a novel homozygous missense variant (NM_001323413.1:c.1867G>A; p.[Asp623Asn]) in exon 15 of the ALDH18A1 was identified. Sanger validation in all available family members confirmed the autosomal recessive modes of inheritances in each family. Three dimensional in‐silico protein modeling suggested deleterious impact of the identified variants. Furthermore, these variants were assigned class 1 or “pathogenic” as per guidelines of American College of Medical Genetics 2015. Screening of ethnically matched healthy controls (n = 200 chromosomes), excluded the presence of these variations in general population.ConclusionsTo the best of our knowledge, this is the first report of ATP6V0A2 and ALDH18A1 variations in the Pakhtun ethnicity of Pakistani population. The study confirms that WES can be used as a first‐line diagnostic test in patients with cutis laxa, and provides basis for population screening and premarital testing to reduce the diseases burden in future generations.