Clinical And Molecular Pathology Study Of Two Genetic Disease Families

Background and AbjectiveGenetic diseases is one of most common diseases coped with in genetic counseling clinic.The main purpose of these patients visting the clinics is:want to know the cause and harm of the genetic diseases;the incidence of filial generation;how to avoind abnormal fetal birthing out.We solve the problem with preimplantation genetic diagnosis（PGD） and prenatal diagnosis（PD）.But we must know the disease genes before performing PGD and PD.So,screening and verifiying disease genes become key works in clinical hospitals.In this paper,two pedigrees of genetic disease were introduced.They are pretibial epidermolysis bullosa （PEB） and the acrofacial dysostoses, AFDs).Materials and Methods1.PEB1.2Materials:Proband IV-3of the family was a23-year-old Chinese woman from the Central region of Guangdong Province who hand suffered with severe itching on the side of pretibial areas since the age of12years. Miliary papules appeared and spreaded after hand scratching.On examination,she showed numerous sharply demarked prurigo nodularis-like skin lesions on pretibial areas of both lower legs.There were also blisters,small round scars and milia.Few lesions were presented on extra-cutaneous.The proband also had toenail fusion. At the age of26-year-old, the symptom had declined on the right lower leg,the lesions on the left lower leg had healed and few milia can be seen. On family line investigation, there are11members affected in four generations. Ⅰ-2, Ⅱ-1, Ⅱ-2and Ⅱ-4had dead,proband’s mother（Ⅲ-3）,mothers’s two sisters（Ⅲ-2,5）,younger brother and two aunt cousins（IV-1,2） all had toenail fusion and a history of blistering on the lower legs. Except for proband and Ⅳ-2,all other members affected had nail dystrophy but only the index finger and thumb of left hand,middle finger,the index finger and thumb of right hand.In addition,the lesions symptom are very different in severity.Proband’s mother（Ⅲ-3） and mother’s older sister（Ⅲ-2） were the most serious.Both presented with erosions,crusts, erythematous patches. Since about6years old,they had suffered from blistering and erosions localized at the knees.following extending anterior tibia and feet. Both of forearms and abdomen of Ⅲ-2presented a few miliary papules but milder,and only on right forearm for Ⅲ-3.Ⅳ-1and IV-4were moderate.In which, IV-1presented miliary papules since7years old. Small atrophic scars existed. And IV-4left leg had no skin（blood leg） when he was born. After healed at6moths, no lesions was on the legs.But at20years old, military papules appeared by scratching for severe itching. Obvious scar were left after trauma healing.Proband, II-5and IV-2were mild. In them, proband was onset from12years old with red papules,blistering and scaring on pretibial aears, and left leg was healed almost at26years old, the lesions was unvisbko Ⅱ-5was milder and the lesions of IV-2lower legs had disappeared almost。1.2Methods:After obtaining informed consent, pedigree was mapped （see Fig.2）and analyzed, clinical features was investigated. SYSMEX-500i cell counter, COB AS800biochemical analyzer and Cobas601Immune analyzer were performed to examine complete cell counting,biochemical and immunological items.Genomic DNA extracted from peripheral blood from the patients and other unaffected family members by the phenol-chloroform extraction method.Genotyping for all available family members were determined using five microsatellite markers（cen-D3S1581, D3S2456, D3S3560, D3S3604-tel）flanking the COL7A1gene. Mutation search was performed by Sanger sequencing. Verification of mutationAgain,sanger sequencing using customized primers was performed to determine the presence of the candidate causative mutation（s） in all available clinically affected subjects and to screen the unaffected members in the family for co-segregation analysis.2. AFDs2.1Materials:The fetus was born in multiple malformation:vaulted eyebrows, inclined and narrow eyelid, split wide apart, blepharoptosis, down eyelid caved in triangular in shape, bilateral outer ear malformation, external canal atresia, nasal convex deformity, low hairline, giant mouth, cheekbones and jaw dysplasia, small jaws deformity, triangle down face form, curve upper extremity, outward elbow, ungula-state hand, and feedthrough palm print, clubfoot, cunnus abnormality, and no limb defects, and refused autopsy. The birth weight was1800g （<3rd）, and the height was45cm （<3rd）. APGAR score was seven points and two points^at lmin and5min respectively. The breathing was supported by expiratory machine, and serious anhelation caused death in six hours after birth.The child’s mother, Lan XX, was Han people, coming from Lingshuijiang county Hunan province China. She was26years old and156cm high with normal phenotype and intelligence. The menarche came at14, cycle by30days, and the period is4-5days, with moderate quantity, no dysmenorrhea, and no clot. The child’s father,30years old, had intelligence and phenotype without special abnormalities and did not have bad habits. Also this couple was not inbreeding marriage. They conceived four times after marriage with once induced abortion and twice natural abortion. And this pregnancy was intrauterine for35w+4, with hydramnios （about4000ml）, blood pressure126/77mmHg, fundus of uterus34cm above the symphysis pubis, the head first exposed, fixed, fetal position LOA, FH140per minute, and ostium uteri4+cm by anus check. The fetal was12days preterm and normally delivered with smooth birth process.2.2Methods:First of all, we made an investigation on family, gestational and medical history and other general information, and collected venous blood to do conventional biological chemistry, immunology and blood cytology check using automatic analyzer. Culturing trace lymphocytes in vitro, chromosomal aberration was excluded by karyotype analysis. Then fluorescence in situ hybridization was used to test TUPLE1gene （lacked in90%patients with congenital heart disease）, ARSA gene （lacked in85%patients with Velocardiofacial syndrome） and DAZ gene （micro missing on the end of chromosomal Y）. After that, comparative genomics hybrid technology was adopted to analyze gene copy number changes. When positive area was found, we referred to PubMed to determine candidate genes which were finally validated by RT-PCR.Results and Discussion1.PEBThe available subjects include7affected and4unaffected individuals.The expressivity of affected individuals was very different. They were divided into severe, moderate and mild subjects respectively. A complete blood cell count, liver and renal function tests, antinuclear and anti-double-stranded DNA antibody titers, and immunoglobulin patterns were normal or negative.Haplotype analysis was performed through four sites selected in COL7A1gene,which showed that all the affected members share the same "semi-haplotype"1-4-1-1inherited from the father which indicates the present disease is related to COL7A1.To screen the mutation, we amplified genomic DNA segments covering all118exons sequences of COL7A1gene by PCR and subjected the products to automated sequencing,which demonstrated a heteroduplex exon73of COL7A1obtained from the proband.This mutant allele demonstrated a heterozygous G-to-T transition at6101（RefSeq Accession:NM₀00094） as shown in figures3A.These nucleotide changes would introduce a substitution of a glycine （GAG） by an valine （GTG） at amino acid position2034in the type Ⅶ collagen.2.AFDsClinical conventional detection showed no abnormalities, as well as number and structure of chromosome. TUPLE1, ARSA and DAZ genes had no deletion. comparative genomics hybrid analysis indicated there were two areas with increased copy number on chromosome1, one of which was located in Ip36.33（between784258bp～1556626bp）, and the length was about722KB. This anomaly was not found in the parents’samples. The segment contained51genes, in which12were known genes,5were macro-RNA, and VMA1gene encoding vWF protein A of extracellular matrix protein super family, which played an important role in cartilage structure and functions. Another area was located in Iq21.3-22（between153182506bp～153318761bp） with the length of136kb. It contained12genes, in which nine were known genes and the PYGO2gene can play an important role in craniofacial bone development by adjusting the Wnt signaling pathways. The results of RT-PCR showed that copy number of the reference gene GAPDH （2-ΔΔCt） was1, but2-ΔΔCt of VWA1and PYGO gene were1.65and2.01respectively, which indicated existing repeat to prove that the results of array-CGH were accurate.Conclusion1.PEB:Anovel heterozygous nucleotide G>T transition at position6101in exon73of COL7A1was detected, which resulted in a glycine to valine substitution （G2034V） in the triple-helical domain of type-VII collagen. Most mutations of the COL7A1gene are family-specific, and families differ with respect to phenotypes. This is the first report to show that one mutation caused a broad range of severity of disease in one family with PEB. These data suggest that c.6101G>T may influence the phenotype of PEB. They also contribute to the expanding database on COL7A1mutations.2.Ads:To investigate the etiology of the phenotype, whole genomic high-resolution array comparative genomic hybridization analysis was carried out, revealing two cryptic duplications,1p36.33and1q21.3-q22duplications. Two genes, VWA1and PYGO2, contained in the two duplications, respectively, are likely to be the candidate genes for the phenotype of our patient.